Oxygen-Dependent Modulation of Insulin-Like Growth Factor Binding Protein Biosynthesis in Primary Cultures of Rat Hepatocytes
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《内分泌学杂志》
Medizinische Klinik und Poliklinik (J.-G.S.), Georg-August-Universitt, D-37075 Gttingen, Germany
Department Biochemistry (T.K.), Faculty of Chemistry, University Kaiserslautern, D-67663 Kaiserslautern, Germany
Departments of Medicine and Physiology and Biophysics (T.G.U.), University of Illinois at Chicago and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612
Abstract
Higher levels of IGF-binding protein 1 (IGFBP-1) mRNA are expressed in the less aerobic perivenous zone of the liver. Because gradients in oxygen tension (pO2) may contribute to zonated gene expression, the influence of arterial and venous pO2 on IGFBP-1 biosynthesis was studied in primary cultures of rat hepatocytes. Maximal IGFBP-1 mRNA and protein levels were observed under venous pO2, whereas less than 30% of maximal levels were observed under arterial pO2. In contrast, the expression of IGFBP-4 was greatest under arterial pO2, indicating that this effect of hypoxia on IGFBP-1 gene expression is specific. The response to hypoxia appears to involve reactive oxygen species, because treatment with H2O2 results in a dose-dependent decrease of IGFBP-1 mRNA levels under venous pO2, whereas IGFBP-1 mRNA expression under arterial pO2 was not affected. Inhibition of the hypoxia-dependent IGFBP-1 mRNA induction by actinomycin D indicates that this effect is mediated at the level of gene transcription, and inhibition of IGFBP-1 mRNA by the iron chelator desferrioxamine under both venous and arterial pO2 suggested the involvement of hypoxia-inducible transcription factors (HIF). Transfection experiments demonstrated that especially HIF-3 and HIF-2, and to a lesser extent HIF-1, contribute to the induction of IGFBP-1 mRNA expression in isolated hepatocytes, whereas experiments with vectors for the HIF prolyl hydroxylases (PHD) indicated a major role of PHD-2 in destabilization of HIFs, attenuating the induction of IGFBP-1 under venous pO2. Reporter gene studies indicate that hypoxia stimulates IGFBP-1 expression through a putative HIF response element located approximately 250 bp upstream from the transcription initiation site. Together, these results support the concept that iron, radical oxygen species, and the HIF-2 and -3 as well as the PHD pathways play important roles in mediating effects of hypoxia on IGFBP-1 gene expression in the liver.
Introduction
IGFs PLAY AN IMPORTANT role in the regulation of metabolism, development, and growth. The capacity of IGFs to exert their biological effects via interaction with specific cell surface receptors is modulated by the presence of a family of structurally related IGF-binding proteins (IGFBPs) (1, 2, 3). So far, six distinct high-affinity IGFBPs have been identified that differ in molecular mass, binding properties for IGFs, and posttranslational modifications as well as tissue and developmental regulated expression. Many functions have been proposed for the IGFBPs, including carrier proteins in the circulation, storage of IGFs in specific tissue compartments, inhibition of IGF action by preventing access to IGF receptors, and the potentiation of the mitogenic response by providing a stable source of available growth factor (1).
In adult rats, the liver has been recognized as the major source of circulating IGF-I and of IGFBP-1 (3). Several studies have indicated that the production of these proteins is distributed between different hepatic cell populations. Rat hepatocytes have been shown to secrete IGFBP-1, -2, and -4, whereas IGFBP-3 is expressed primarily by nonparenchymal liver cells (4). In situ hybridization studies using an IGFBP-1 riboprobe revealed that IGFBP-1 mRNA expression is highest in the less aerobic perivenous region of the hepatic acinus and that transcript levels are reduced in the more aerobic periportal area (5). Zonated gene expression also has been demonstrated for a number of gene products that are involved in carbohydrate, lipid, and xenobiotic metabolism or bile formation and hepatobiliary transport of bile constituents (6, 7, 8, 9, 10).
Although a number of different gradients exist within the liver acinus, oxygen tension (pO2), which decreases by about 50% from the periportal to the perivenous regions, has been considered to be a key regulator for zonated gene expression. However, the O2 signaling pathways mediating this effect have not been fully characterized. The O2 sensing process appears to involve multiple factors, including oxygen-binding hemeproteins, the generation of reactive oxygen species (11, 12), oxygen-dependent prolyl (13, 14, 15, 16) and asparaginyl hydroxylases (17, 18), and the -subunit hypoxia-inducible transcription factors (HIFs), which play a central role in O2-regulated gene expression.
HIFs are heterodimers consisting of an -subunit (HIF-1, HIF-2, and HIF-3) and -subunits that belong to the aryl hydrocarbon receptor nuclear translocator (ARNT) family (ARNT, ARNT2, and ARNT3). Transactivation of genes under hypoxia involves dimerization of the two HIF subunits, which bind to an enhancer element called the hypoxia-response element (HRE) in target genes (19, 20). Under high pO2 conditions, hydroxylation of at least two proline residues promotes the degradation of HIF-1 -subunits, and this process is carried out by a family of newly identified prolyl hydroxylases (PHDs), including PHD-1, PHD-2, PHD-3, and PHD-4 (13, 14, 15, 16, 21). Hydroxylation enables the binding of the von Hippel-Lindau tumor suppressor protein (pVHL), a component of an E3 ubiquitin ligase complex that targets the HIF -subunits for degradation by the ubiquitin-proteasome pathway (22). In a similar manner, the hydroxylation of an asparaginyl residue in the C-terminal transactivation domain of HIF-1 and HIF-2 prevents the recruitment of the coactivator CBP/p300, thus reducing the HIF-1 and HIF-2 transactivation potential (18, 23, 24).
The aim of the present study was to investigate whether the O2 tension may be a modulator of IGFBP expression in primary hepatocytes cultured under arterial (16% O2) and venous (8% O2) pO2. Initial experiments demonstrated that expression of IGFBP-1 was induced under venous pO2, whereas the expression of IGFBP-4 was highest under arterial pO2, indicating that these effects are specific and that rat hepatocytes provide an excellent cell model for investigating mechanisms of differential IGFBP gene expression under arterial vs. venous conditions. We examined the roles of iron and reactive oxygen species in the oxygen-sensing process regulating IGFBP expression by means of the iron chelator desferrioxamine (DSF) and by exposure of rat hepatocytes to exogenous H2O2. The involvement of HIFs and of PHDs in O2-dependent IGFBP regulation was evaluated by transfection of rat hepatocytes with expression vectors for HIF-1, HIF-2, and HIF-3 as well as PHD-1, PHD-2, PHD-3, and PHD-4 and subsequent cultivation of transfected cells under arterial and venous pO2. Transient transfection studies with reporter gene constructs indicate that hypoxia stimulates the activity of the IGFBP-1 promoter in isolated hepatocytes and that mutation of the HRE disrupts this effect.
Materials and Methods
Animals
Male Wistar rats (200–260 g) were kept on a 12-h day/night rhythm (light from 0700–1900 h) with free access to water and food. Rats were anesthetized with pentobarbital (60 mg/kg body weight) before preparation of hepatocytes between 0800 and 0900 h. The study protocols were approved by a government review board. All animals received care in compliance with institutional guidelines, the German Convention for Protection of Animals and the National Institutes of Health guidelines.
Cell culture
Hepatocytes were isolated by collagenase perfusion. Cells (1 x 106 per dish) were maintained under standard conditions in a normoxic atmosphere of 16% O2, 79% N2, and 5% CO2 (by volume) in medium M199 containing 0.5 nM insulin added as a growth factor for culture maintenance, 100 nM dexamethasone required as a permissive hormone, and 4% fetal calf serum for the initial 4 h of culture. Cells were then cultured in serum-free medium until 24 h at 16% O2. At 24 h, medium was changed and culture was continued at normoxia (16% O2) or at mild hypoxia [8% O2, 87% N2, 5% CO2 (by volume)]. Sixteen percent O2 and 8% O2 at the medium surface correspond to 13% O2 (arterial pO2) and 6% O2 (venous pO2) at the cell surface (25). In actinomycin experiments, hepatocytes were pretreated for 30 min with actinomycin D (ActD) (1 μg/ml) before the cells were cultured under normoxia or mild hypoxia for 6–12 h. Desferrioxamine was from Sigma Chemical Co. (St. Louis, MO), and cells were treated as indicated with 130 μM for 12 h.
Plasmids and cDNA probes
cDNA probes were used for Northern blot analysis, including a 407-bp fragment of rat IGFBP-1 cDNA clone pRBP-1-501 (26) and a 444-bp fragment of rat IGFBP-4 cDNA clone pRBP4-SH (26). As a control to quantify Northern blots, an oligonucleotide (5'-AAC GAT CAG AGT AGT GGT ATT TCA CC-3') complementary to 28 S rRNA was used as a hybridization probe. The expression plasmids containing the full-length rat HIF-1, HIF-2, and HIF-3 cDNA under the control of the cytomegalovirus promoter, the erythropoietin (EPO)-HRE construct, its mutant, and the IGFBP-1 promoter construct have already been described (27, 28, 29). The mutant IGFBP-1 promoter was generated by site-directed mutagenesis using the QuikChange mutagenesis kit (Promega, Madison, WI). The V5-tagged PHD-1, -3, and -4 expression vectors were generated after amplification of the respective PHD cDNA fragment by PCR with HepG2 cell cDNA as the template. Primers used were the following: forward 5'-CACCATGGACAGCCCGTGCCAGCCG-3', reverse 5'-ggtgggcgtaggcggctgtgatacag-3' for PHD1; forward 5'-CACCGCCATGGCCAATGACAGC-3', reverse 5'-gaagacgtctttaccgaccgaatct-3' for PHD2; forward 5'-CACCGAGATGCCCCTGGGACACATCATGAGGCT-3', reverse 5'-ctgcaggaattcaggaaccggtcttcagtgagggcagatt-3' for PHD3; and forward 5'-CACCATGGCGGCAGCGGCGGTGACAG-3', reverse 5'-gagttccacgcgcgcatcgcggtaggc-3' for PHD4. PCR fragments were cloned into pCR2-TOPO/V5 (Invitrogen GmbH, Karlsruhe, Germany) according to the instructions of the manufacturer. The PHD-2 vector was generated in a similar way but with an already cloned PHD-2 cDNA as template (15). All constructs were verified by sequencing in both directions.
Cell transfection and luciferase assay
Freshly isolated rat hepatocytes (about 1 x 106 cells per dish) were transfected for 5 h with 2.5 μg plasmid DNA essentially as described (30). After 5 h, the medium was changed and the cells were cultured under normoxia for 19 h. Then, medium was changed again and the cells were additionally cultured for 24 h under arterial or venous pO2.
Ligand and Western blotting
Ligand blotting of conditioned media (CM) from primary hepatocytes using [125I]IGF-I as tracer was performed according to Hossenlopp et al. (31) with slight modifications as described recently (32, 33). Western blot analysis was carried out as described (30). The primary antibody against HIF-1, HIF-2, and HIF-3 were described earlier (27) and used in a 1:800 dilution. The primary monoclonal antibody against the V5-Tag was from Invitrogen and used in a 1:1000 dilution. Bound antibodies were detected by a goat antirabbit IgG-horseradish peroxidase (Santa Cruz Biotechnology, Heidelberg, Germany) or an antimouse IgG-horseradish peroxidase, respectively, used in a 1:2000 dilution. The ECL Western blotting system (Amersham Pharmacia Biotech, Braunschweig, Germany) was used for detection.
Isolation of total RNA and Northern blot analysis
Total RNA was isolated from rat liver cells as described recently (4, 34). For IGFBP-1, total RNA was evaluated using digoxigenin-labeled antisense RNAs generated by in vitro transcription with RNA polymerase (Roche, Mannheim, Germany) and the T3 promoter of pBluescript SK+ plasmid as described previously (33). Fragments of the rat IGFBP-4 plasmid (444 bp) were [32P]dCTP labeled by random priming. Prehybridization, hybridization, and washing of membranes were performed essentially as described (4, 34).
Statistical analysis
Autoradiographs of ligand and Northern blots were scanned (Gelcam Phase, Lübeck, Germany) and densitometrically analyzed (Molecular Analyst; Bio-Rad, Hercules, CA). The relative densities of bands were expressed as the percent increase or decrease compared with the respective untreated control and indicated as mean ± SD. The Student's t test for paired values with a P value of <0.05 was considered significant.
Results
Time course of O2-modulated expression of IGFBP-1 and IGFBP-4 mRNA and protein
To evaluate the effects of pO2 on IGFBP mRNA expression, rat hepatocytes were preincubated in serum-free medium for 1 h and then cultured in fresh serum-free medium under arterial (16% O2) or venous (8% O2) conditions for 6, 12, and 24 h. Under hypoxic conditions, IGFBP-1 mRNA abundance increased rapidly and reached maximal levels after 12 h of incubation (Fig. 1). In contrast, steady-state IGFBP-4 mRNA expression was stimulated under arterial pO2 with maximal levels after 12 h, whereas under venous pO2, IGFBP-4 mRNA remained at basal levels throughout the 24 h of culture (Fig. 1).
We evaluated the secretion of IGFBP proteins into CM of hepatocytes under arterial and venous conditions by [125I]IGF-I ligand blotting (Fig. 2) and Western blotting (data not shown). An increase in IGFBP-1 protein levels was detected after 6 h of culture under hypoxic conditions, and this effect persisted at all time points tested. After 12 h exposure to hypoxia, the amount of IGFBP-1 protein was increased 2-fold (Fig. 2). In contrast, maximal IGFBP-4 protein levels were detected under arterial pO2. After 24 h of culture, IGFBP-4 protein under venous pO2 was reduced by 35% compared with levels observed under arterial pO2 (Fig. 2).
To determine whether hypoxia regulates IGFBP-1 expression at the transcriptional level, rat hepatocytes were treated with the transcriptional inhibitor ActD (1 μg/ml) for 30 min and then placed under 16 or 8% pO2 for 6 and 12 h. Addition of ActD prevented the hypoxia-induced increase in IGFBP-1 mRNA at 6 h (Fig. 3) and reduced the secretion of IGFBP-1 protein after 12 h of cultivation under both arterial and venous conditions (Fig. 4) consistent with transcriptional regulation. ActD treatment of hepatocytes also prevented the normoxia-dependent induction of IGFBP-4 mRNA (Fig. 3) and IGFBP-4 protein (Fig. 4), although to a lesser extent than that observed for IGFBP-1.
Inhibition by H2O2 and enhancement by DSF of the venous pO2-dependent induction of IGFBP-1 mRNA
Iron- and redox-dependent processes have been shown to be involved in oxygen-regulated gene expression (35, 36, 37, 38, 39). We examined the effect of H2O2 (a generator of reactive oxygen species) on IGFBP-1 mRNA expression under arterial and venous pO2. H2O2 dose-dependently decreased IGFBP-1 mRNA levels under venous pO2, whereas IGFBP-1 mRNA expression under arterial pO2 was not affected by addition of H2O2 (Fig. 5).
The participation of iron in oxygen sensing was examined by treatment of rat hepatocytes with DSF, an iron chelator and inhibitor of heme synthesis with antioxidant properties. In the presence of 130 μM DSF, steady-state levels of IGFBP-1 mRNA were increased compared with the untreated control under both venous and arterial pO2 (Fig. 5).
Thus, H2O2 treatment simulated the effect of arterial pO2 and decreased the expression of IGFBP-1 mRNA under venous pO2, suggesting that reactive oxygen species may contribute to the effects of differences in oxygen tension on the expression of IGFBP-1. In contrast, chelation of iron caused by addition of DSF mimicked the effect of venous pO2 and further potentiated the induction of IGFBP-1 mRNA under both arterial and venous pO2, suggesting that iron may be important for O2-dependent IGFBP regulation.
HIF-3 contributes to the hypoxia-dependent induction of IGFBP-1 mRNA
HIFs have been shown to activate a number of genes under low O2 tension. To test whether HIFs activate IGFBP-1 expression in primary rat hepatocytes, we performed transient transfection studies with expression vectors for HIF-1, HIF-2, and HIF-3 and verified the expression of HIF-1, HIF-2, and HIF-3 by Western blotting (Fig. 6). Transfection experiments showed a HIF--dependent increase in IGFBP-1 mRNA expression under both venous and arterial pO2 when compared with the controls transfected with the empty expression vector. Interestingly, the most pronounced effects were observed in hepatocytes transfected with HIF-3, showing approximately 3-fold induction of IGFBP-1 mRNA expression under arterial pO2 and approximately 2-fold induction under venous pO2, respectively (Fig. 6). The expression of IGFBP-1 mRNA was increased by approximately 2-fold under arterial pO2 and approximately 1.5-fold under venous pO2 in cells transfected with the HIF-2 expression vector compared with the appropriate controls. Only a mild stimulation of IGFBP-1 mRNA expression was detectable after transfection with HIF-1 (Fig. 6). In contrast, transfection of hepatocytes with the different -subunits of HIF diminished the induction of IGFBP-4 mRNA under arterial pO2. Again, the stronger effects were found with HIF-3 and HIF-2 where transfection with HIF-2 and HIF-3 reduced the steady-state IGFBP-4 mRNA to about 65 and 45% of control levels under arterial pO2, whereas transfection with HIF-1 resulted only in a more modest decrease of IGFBP-4 mRNA abundance (Fig. 6). Similar alterations as observed at the IGFBP mRNA level after transfection with the different HIF vectors were also detected at the protein level (Fig. 7).
PHD-2 attenuates the hypoxia-dependent induction of IGFBP-1 mRNA
At normal oxygen tension, PHDs hydroxylate specific proline residues in the oxygen-dependent degradation domain of HIF -subunits to allow their binding to pVHL, which targets prolyl-hydroxylated HIF -subunits for degradation by the ubiquitin-proteasome system. To elucidate the role of PHDs in the regulation of hepatic IGFBP expression under normoxia and hypoxia, primary rat hepatocytes were transfected with expression vectors for PHD-1, PHD-2, PHD-3, and PHD-4 and subsequently cultivated under arterial and venous pO2. Expression of PHDs in hepatocytes was evaluated by Western blotting using anti-V5-Tag antibodies showing protein bands for PHD-1 and -2 at 44 and 46 kDa, respectively, for PHD-3 at 27 kDa and for PHD-4 at 56 and 50 kDa (Fig. 8). In addition, the effects of PHD transfections were also investigated on the level of the HIF -subunits. It was found that in PHD-2-transfected cells all three HIF -subunits were barely detectable, whereas PHD-3 was less effective but also decreasing the level of all three HIF -subunits, although a hypoxia-dependent induction was still apparent (Fig. 9). Transfection with PHD-1 and PHD-4 expression vectors only modestly reduced the level of HIF-1 under hypoxic conditions, whereas the effect of hypoxia on the expression of HIF-2 and HIF-3 was nearly abolished (Fig. 9).
Furthermore, transfection of hepatocytes with PHD-1, PHD-2, PHD-3, and PHD-4 consistently attenuated the induction of IGFBP-1 mRNA under venous conditions (Fig. 8). Although PHD-1 and PHD-4 transfection mediated only a slight decrease, PHD-2 and PHD-3 reduced IGFBP-1 mRNA levels to about 70 and 60%, respectively, of maximal levels observed in vector transfected hepatocytes under venous pO2.
Transfection with PHD vectors also altered the effect of arterial pO2 on the expression of IGFBP-4 mRNA. Transfection of PHD-1 decreased IGFBP-4 mRNA levels by approximately 30% under arterial pO2, whereas IGFBP-4 mRNA abundance was not significantly altered under venous conditions. Furthermore, PHD-2 reduced IGFBP-4 mRNA abundance by approximately 50% under arterial pO2 and about 20% under venous pO2. PHD-3 had an even more profound effect; decreasing IGFBP-4 mRNA levels by 70 and 85%, respectively. The transfection of PHD-4 had no effect on the IGFBP-4 mRNA levels (Fig. 8). The observed changes in IGFBP-1 mRNA levels after transfection with PHD vectors were reflected by changes in the levels of IGFBP proteins released into hepatocyte-CM (Fig. 9).
Together, these results indicate that IGFBP-1 and -4 expression in hepatocytes can be modulated via the HIF and PHD pathways with HIF-3 and PHD-2 playing a major role in this setting.
The sequence –252/–247 contributes to hypoxia-dependent induction of IGFBP-1
To test whether hypoxia exerts an effect on the IGFBP-1 promoter, we transfected an luciferase (Luc) reporter construct driven by the first 320 bp of the rat IGFBP-1 promoter. We found that venous conditions induced Luc activity by about 2-fold. Similarly, hypoxia mediated a 2-fold increase in Luc activity after transfection of a construct containing three HREs from the erythropoietin gene in front of the SV40 promoter. We next searched for the presence of the HRE consensus sequence within the rat IGFBP-1 promoter, and only the sequence 5'-CAAGTG-3' at –252/–247 resembled a 5'-RCGTG-3' HRE consensus sequence with one mismatch. Mutation of this sequence within the –320 IGFBP-1 promoter Luc construct abolished induction by hypoxic conditions. Likewise, mutation of the HIF-binding EPO-HRE also abolished induction by hypoxia (Fig. 10). Thus, the present data suggest that the sequence –252/–247 contributes to the hypoxia-dependent IGFBP-1 induction via binding of HIFs.
Discussion
The present study demonstrates that reduced oxygen tension enhances IGFBP-1 mRNA and protein expression in primary cultures of rat hepatocytes. In contrast, the expression of IGFBP-4 mRNA and protein are reduced by low pO2 conditions. The positive modulation of IGFBP-1 expression by venous pO2 was further stimulated by the iron chelator DSF, whereas H2O2 decreased IGFBP-1 mRNA levels, supporting the concept that iron and reactive oxygen species are involved in O2 sensing and the regulation of IGFBP-1. Transient transfection experiments demonstrated an involvement of HIF-3 and HIF-2 and to a lesser extent of HIF-1 for the induction of IGFBP-1 mRNA under venous conditions. The involvement of HIFs is further corroborated by experiments showing a role of PHDs in destabilization of HIFs, thereby attenuating the modulation by O2 of IGFBP-1 and -4 expression.
Oxygen as modulator of zonated gene expression
The results of the present study suggest that reduced pO2 in the perivenous region may constitute a critical determinant for the zonated expression of IGFBP-1 in rat liver. This is in line with findings indicating that hypoxia and the IGF system are interrelated (40). In vivo studies of rats fed with normal and high-protein diets showed higher levels of IGFBP-1 mRNA in the perivenous compared with the periportal zone of the liver (5). In addition, animal models of uteroplacental insufficiency (caused by uterine artery ligation or maternal hypoxia) resulted in fetal hypoxia and intrauterine growth restriction, with marked elevated circulating levels of IGFBP-1 and overexpression of IGFBP-1 mRNA in the fetal liver, the primary source of IGFBP-1 (40, 41, 42, 43). Furthermore, hypoxia stimulated IGFBP-1 expression in human HepG2 hepatoma cells as well as in primary cultures of human fetal hepatocytes (44, 45, 46).
Similar to IGFBP-1, the expression of glucokinase is greatest in the perivenous region of the hepatic acinus, and reduced pO2 stimulates the expression of glucokinase in isolated rat hepatocytes (47). In contrast, in this study we find that arterial pO2 is associated with increased expression of IGFBP-4 compared with low pO2. This is similar to previous results obtained with periportally expressed genes, including phosphoenolpyruvate carboxykinase (48, 49) and tyrosine aminotransferase (25).
In addition to the periportal to perivenous gradient of pO2 as a key regulator of the zonated gene expression, it also may be speculated that the gradient in IGFBP-1 expression is partly a result of changes in insulin levels across the liver acinus. Although up to 50% of insulin is degraded from the periportal to the perivenous zones (9), insulin receptor expression is higher in the perivenous zone (50), supporting the concept that the perivenous zone also is an important site of insulin action in the liver. By contrast, the glucagon receptor has been shown to be expressed predominantly in the periportal zone of the liver acinus (51). Because insulin is inhibitory (33) and glucagon is stimulatory (52, 53) to hepatic IGFBP-1 expression, the effects of insulin and glucagon are not likely to represent the underlying mechanism for the perivenous localization of IGFBP-1 expression in the liver.
Involvement of reactive oxygen species in O2 signaling
Reactive oxygen species derived from H2O2 have been proposed to be messengers of the O2 signal in hepatoma cells (36) as well as in hepatocytes (54). H2O2 is a highly diffusible molecule that is an ideal candidate for functioning as a second messenger that, in the presence of iron, yields highly reactive hydroxyl radicals via a local Fenton reaction (55). These radicals may lead to oxidation of proteins, thereby altering the function or stability of biologically active proteins, including transcription factors. H2O2 might mimic the effect of arterial pO2 by destabilizing the -subunit of the transcription factor HIF-1 and HIF-2 (56, 57). However, other studies report that HIF levels are enhanced in the presence of H2O2 (58, 59). In the present study, H2O2 decreased the induction of IGFBP-1 mRNA expression under venous pO2, thereby supporting the initial findings that H2O2 mimics the effect of arterial pO2, similar to its effects on the expression of PEPCK mRNA in primary hepatocytes (49, 55). Reciprocally, GK gene expression was down-regulated by H2O2 (47, 55). Thus, repression of hypoxia-dependent enhancement of IGFBP-1 mRNA by H2O2 further strengthens the proposal that H2O2 might function as a second messenger in regulation of IGFBP-1 expression by oxygen.
HIFs and HIF-prolyl hydroxylases
The oxygen-signaling system controlling induction of a variety of genes by hypoxia is present in most if not all cells and involves the induction of HIFs, which bind to HREs located in either the 5' or the 3' regions of the genes. Studies in human hepatoma cells have shown that HIF-1 can stimulate the expression of IGFBP-1 (44); however, the role of other HIFs such as HIF-2 or HIF-3 was not studied. The results of the present study suggest that the induction of IGFBP-1 by venous pO2 is mediated predominantly by HIF-3 and -2 and only to a lesser extent via HIF-1 in primary cultures of rat hepatocytes.
Under normoxia, HIF -subunit destabilization is mediated by O2-dependent hydroxylation of at least two proline residues within the oxygen-dependent degradation domain (13, 14, 15, 21, 60). The pVHL E3 ubiquitin ligase complex associates with hydroxylated proline residues and targets HIF -subunits for proteasomal degradation. Under hypoxia, oxygen becomes rate limiting for the at least four proline hydroxylase enzymes (PHDs) (15, 61). As a consequence, HIF -subunits accumulate, migrate into the nucleus, and associate with ARNT, and this complex interacts with HREs of target genes (60, 62). The results of the present study indicate a major role of PHD-2 in the destabilization of HIFs and reduced expression of IGFBP-1 under normoxic conditions in isolated hepatocytes. The effect of PHD-1, PHD-3, and PHD-4 was less prominent. These findings are largely consistent with another recent study showing that in Hela cells, PHD-2 was the major functional HIF-modifying enzyme, whereas PHD-1 and PHD-3 were ineffective, and PHD-4 was not investigated (63). The variation with respect to the action of PHD-3 may be eventually explained by cell-type-specific differences. Of note, because PHDs require Fe2+ in addition to O2, the induction of IGFBP-1 after treatment with DSF as observed in the present study might be a result of impairment of PHD activity, consistent with other studies (14, 15).
Interestingly, our study also showed that IGFBP-4 expression is repressed under hypoxia compared with normoxia. It might be speculated that higher gene expression under normoxia as shown in this study could be a result of an inhibitory effect of HIFs under hypoxia. The cotransfection experiments with the HIF expression vectors would support this because the normoxic IGFBP-4 levels are down-regulated after HIF -subunit transfection. Although HIFs act usually as activators, the role of HIFs as transcriptional inhibitors was also demonstrated in another study with the peroxisome proliferator-activated receptor- gene (64).
By contrast, the experiments using the PHD expression vectors do not completely support this concept because overexpression of PHD-1, -2, and -3 attenuated normoxic IGFBP-4 expression. This could be explained by the additional action of a so far unknown IGFBP-4 inducer that could be degraded by the action of PHDs.
Multiple regulatory elements have been identified in the IGFBP-1 promoter (65), including an insulin response element that mediates the negative effect of insulin on basal IGFBP-1 promoter activity and two glucocorticoid response elements. In addition, three consensus HREs have been identified in intron 1 of the human IGFBP-1 gene, including at least one that is hypoxia responsive with regard to IGFBP-1 gene expression. Recent studies suggest that other HREs located approximately 1000 bp upstream of the transcription initiation site may contribute to the regulation of IGFBP-1 in zebrafish and possibly other species (Kajimura, S., and C. Duan, personal communication). In the present study, we identified an HRE located approximately 250 bp upstream from the transcription initiation site in the rat IGFBP-1 promoter based on its relationship to the consensus HRE sequence. Functional studies confirmed that this element is necessary for the ability of hypoxia to stimulate IGFBP-1 promoter activity. This element is highly conserved in mouse and rat and partially conserved in the human IGFBP-1 promoter, and the ability of this site to bind and mediate effects of different HIFs on IGFBP-1 promoter activity is currently under investigation. Interestingly, this HRE resides in a region that has been reported to be important in enhancing effects of glucocorticoids on IGFBP-1 promoter activity (66), suggesting the interesting possibility that the interactions with this element may contribute cooperatively to the regulation of IGFBP-1 by other factors. Additional studies are required to unravel the role of this and other HREs in mediating the effects of hypoxia and other factors on IGFBP-1 expression. Additional studies regarding the mechanisms mediating the effects of high and low pO2 on the expression of IGFBP-4 also will be of significant interest.
In conclusion, the present study demonstrates that hypoxia enhances IGFBP-1 and reduces IGFBP-4 expression in primary rat hepatocytes. These studies provide additional evidence for the involvement of an oxygen-radical-generating process and the HIF system in oxygen sensing and in the regulation of zonated gene expression in the liver.
Acknowledgments
The technical assistance of Sieglinde Zachmann, Susanne Hupe, and Katja Curth is gratefully acknowledged.
Footnotes
This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 402, project A1 (to T.K.), GRK 335/2 (to T.K., J.-G.S.), and Scha 700/1-2 (to J.-G.S.); Georg-August-Universitt Gttingen, Schwerpunktfrderung Onkologie (to J.-G.S.), National Institutes of Health Grant DK41430 (to T.G.U.); and Department of Veterans Affairs Merit Review Program (to T.G.U.).
First Published Online September 15, 2005
Abbreviations: ActD, Actinomycin D; ARNT, aryl hydrocarbon receptor nuclear translocator; CM, conditioned medium; DSF, desferrioxamine; EPO, erythropoietin; HIF, hypoxia-inducible transcription factor; HRE, hypoxia-response element; IGFBP, IGF-binding protein; PHD, prolyl hydroxylase; pVHL, von Hippel-Lindau tumor suppressor protein.
Accepted for publication September 2, 2005.
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Department Biochemistry (T.K.), Faculty of Chemistry, University Kaiserslautern, D-67663 Kaiserslautern, Germany
Departments of Medicine and Physiology and Biophysics (T.G.U.), University of Illinois at Chicago and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612
Abstract
Higher levels of IGF-binding protein 1 (IGFBP-1) mRNA are expressed in the less aerobic perivenous zone of the liver. Because gradients in oxygen tension (pO2) may contribute to zonated gene expression, the influence of arterial and venous pO2 on IGFBP-1 biosynthesis was studied in primary cultures of rat hepatocytes. Maximal IGFBP-1 mRNA and protein levels were observed under venous pO2, whereas less than 30% of maximal levels were observed under arterial pO2. In contrast, the expression of IGFBP-4 was greatest under arterial pO2, indicating that this effect of hypoxia on IGFBP-1 gene expression is specific. The response to hypoxia appears to involve reactive oxygen species, because treatment with H2O2 results in a dose-dependent decrease of IGFBP-1 mRNA levels under venous pO2, whereas IGFBP-1 mRNA expression under arterial pO2 was not affected. Inhibition of the hypoxia-dependent IGFBP-1 mRNA induction by actinomycin D indicates that this effect is mediated at the level of gene transcription, and inhibition of IGFBP-1 mRNA by the iron chelator desferrioxamine under both venous and arterial pO2 suggested the involvement of hypoxia-inducible transcription factors (HIF). Transfection experiments demonstrated that especially HIF-3 and HIF-2, and to a lesser extent HIF-1, contribute to the induction of IGFBP-1 mRNA expression in isolated hepatocytes, whereas experiments with vectors for the HIF prolyl hydroxylases (PHD) indicated a major role of PHD-2 in destabilization of HIFs, attenuating the induction of IGFBP-1 under venous pO2. Reporter gene studies indicate that hypoxia stimulates IGFBP-1 expression through a putative HIF response element located approximately 250 bp upstream from the transcription initiation site. Together, these results support the concept that iron, radical oxygen species, and the HIF-2 and -3 as well as the PHD pathways play important roles in mediating effects of hypoxia on IGFBP-1 gene expression in the liver.
Introduction
IGFs PLAY AN IMPORTANT role in the regulation of metabolism, development, and growth. The capacity of IGFs to exert their biological effects via interaction with specific cell surface receptors is modulated by the presence of a family of structurally related IGF-binding proteins (IGFBPs) (1, 2, 3). So far, six distinct high-affinity IGFBPs have been identified that differ in molecular mass, binding properties for IGFs, and posttranslational modifications as well as tissue and developmental regulated expression. Many functions have been proposed for the IGFBPs, including carrier proteins in the circulation, storage of IGFs in specific tissue compartments, inhibition of IGF action by preventing access to IGF receptors, and the potentiation of the mitogenic response by providing a stable source of available growth factor (1).
In adult rats, the liver has been recognized as the major source of circulating IGF-I and of IGFBP-1 (3). Several studies have indicated that the production of these proteins is distributed between different hepatic cell populations. Rat hepatocytes have been shown to secrete IGFBP-1, -2, and -4, whereas IGFBP-3 is expressed primarily by nonparenchymal liver cells (4). In situ hybridization studies using an IGFBP-1 riboprobe revealed that IGFBP-1 mRNA expression is highest in the less aerobic perivenous region of the hepatic acinus and that transcript levels are reduced in the more aerobic periportal area (5). Zonated gene expression also has been demonstrated for a number of gene products that are involved in carbohydrate, lipid, and xenobiotic metabolism or bile formation and hepatobiliary transport of bile constituents (6, 7, 8, 9, 10).
Although a number of different gradients exist within the liver acinus, oxygen tension (pO2), which decreases by about 50% from the periportal to the perivenous regions, has been considered to be a key regulator for zonated gene expression. However, the O2 signaling pathways mediating this effect have not been fully characterized. The O2 sensing process appears to involve multiple factors, including oxygen-binding hemeproteins, the generation of reactive oxygen species (11, 12), oxygen-dependent prolyl (13, 14, 15, 16) and asparaginyl hydroxylases (17, 18), and the -subunit hypoxia-inducible transcription factors (HIFs), which play a central role in O2-regulated gene expression.
HIFs are heterodimers consisting of an -subunit (HIF-1, HIF-2, and HIF-3) and -subunits that belong to the aryl hydrocarbon receptor nuclear translocator (ARNT) family (ARNT, ARNT2, and ARNT3). Transactivation of genes under hypoxia involves dimerization of the two HIF subunits, which bind to an enhancer element called the hypoxia-response element (HRE) in target genes (19, 20). Under high pO2 conditions, hydroxylation of at least two proline residues promotes the degradation of HIF-1 -subunits, and this process is carried out by a family of newly identified prolyl hydroxylases (PHDs), including PHD-1, PHD-2, PHD-3, and PHD-4 (13, 14, 15, 16, 21). Hydroxylation enables the binding of the von Hippel-Lindau tumor suppressor protein (pVHL), a component of an E3 ubiquitin ligase complex that targets the HIF -subunits for degradation by the ubiquitin-proteasome pathway (22). In a similar manner, the hydroxylation of an asparaginyl residue in the C-terminal transactivation domain of HIF-1 and HIF-2 prevents the recruitment of the coactivator CBP/p300, thus reducing the HIF-1 and HIF-2 transactivation potential (18, 23, 24).
The aim of the present study was to investigate whether the O2 tension may be a modulator of IGFBP expression in primary hepatocytes cultured under arterial (16% O2) and venous (8% O2) pO2. Initial experiments demonstrated that expression of IGFBP-1 was induced under venous pO2, whereas the expression of IGFBP-4 was highest under arterial pO2, indicating that these effects are specific and that rat hepatocytes provide an excellent cell model for investigating mechanisms of differential IGFBP gene expression under arterial vs. venous conditions. We examined the roles of iron and reactive oxygen species in the oxygen-sensing process regulating IGFBP expression by means of the iron chelator desferrioxamine (DSF) and by exposure of rat hepatocytes to exogenous H2O2. The involvement of HIFs and of PHDs in O2-dependent IGFBP regulation was evaluated by transfection of rat hepatocytes with expression vectors for HIF-1, HIF-2, and HIF-3 as well as PHD-1, PHD-2, PHD-3, and PHD-4 and subsequent cultivation of transfected cells under arterial and venous pO2. Transient transfection studies with reporter gene constructs indicate that hypoxia stimulates the activity of the IGFBP-1 promoter in isolated hepatocytes and that mutation of the HRE disrupts this effect.
Materials and Methods
Animals
Male Wistar rats (200–260 g) were kept on a 12-h day/night rhythm (light from 0700–1900 h) with free access to water and food. Rats were anesthetized with pentobarbital (60 mg/kg body weight) before preparation of hepatocytes between 0800 and 0900 h. The study protocols were approved by a government review board. All animals received care in compliance with institutional guidelines, the German Convention for Protection of Animals and the National Institutes of Health guidelines.
Cell culture
Hepatocytes were isolated by collagenase perfusion. Cells (1 x 106 per dish) were maintained under standard conditions in a normoxic atmosphere of 16% O2, 79% N2, and 5% CO2 (by volume) in medium M199 containing 0.5 nM insulin added as a growth factor for culture maintenance, 100 nM dexamethasone required as a permissive hormone, and 4% fetal calf serum for the initial 4 h of culture. Cells were then cultured in serum-free medium until 24 h at 16% O2. At 24 h, medium was changed and culture was continued at normoxia (16% O2) or at mild hypoxia [8% O2, 87% N2, 5% CO2 (by volume)]. Sixteen percent O2 and 8% O2 at the medium surface correspond to 13% O2 (arterial pO2) and 6% O2 (venous pO2) at the cell surface (25). In actinomycin experiments, hepatocytes were pretreated for 30 min with actinomycin D (ActD) (1 μg/ml) before the cells were cultured under normoxia or mild hypoxia for 6–12 h. Desferrioxamine was from Sigma Chemical Co. (St. Louis, MO), and cells were treated as indicated with 130 μM for 12 h.
Plasmids and cDNA probes
cDNA probes were used for Northern blot analysis, including a 407-bp fragment of rat IGFBP-1 cDNA clone pRBP-1-501 (26) and a 444-bp fragment of rat IGFBP-4 cDNA clone pRBP4-SH (26). As a control to quantify Northern blots, an oligonucleotide (5'-AAC GAT CAG AGT AGT GGT ATT TCA CC-3') complementary to 28 S rRNA was used as a hybridization probe. The expression plasmids containing the full-length rat HIF-1, HIF-2, and HIF-3 cDNA under the control of the cytomegalovirus promoter, the erythropoietin (EPO)-HRE construct, its mutant, and the IGFBP-1 promoter construct have already been described (27, 28, 29). The mutant IGFBP-1 promoter was generated by site-directed mutagenesis using the QuikChange mutagenesis kit (Promega, Madison, WI). The V5-tagged PHD-1, -3, and -4 expression vectors were generated after amplification of the respective PHD cDNA fragment by PCR with HepG2 cell cDNA as the template. Primers used were the following: forward 5'-CACCATGGACAGCCCGTGCCAGCCG-3', reverse 5'-ggtgggcgtaggcggctgtgatacag-3' for PHD1; forward 5'-CACCGCCATGGCCAATGACAGC-3', reverse 5'-gaagacgtctttaccgaccgaatct-3' for PHD2; forward 5'-CACCGAGATGCCCCTGGGACACATCATGAGGCT-3', reverse 5'-ctgcaggaattcaggaaccggtcttcagtgagggcagatt-3' for PHD3; and forward 5'-CACCATGGCGGCAGCGGCGGTGACAG-3', reverse 5'-gagttccacgcgcgcatcgcggtaggc-3' for PHD4. PCR fragments were cloned into pCR2-TOPO/V5 (Invitrogen GmbH, Karlsruhe, Germany) according to the instructions of the manufacturer. The PHD-2 vector was generated in a similar way but with an already cloned PHD-2 cDNA as template (15). All constructs were verified by sequencing in both directions.
Cell transfection and luciferase assay
Freshly isolated rat hepatocytes (about 1 x 106 cells per dish) were transfected for 5 h with 2.5 μg plasmid DNA essentially as described (30). After 5 h, the medium was changed and the cells were cultured under normoxia for 19 h. Then, medium was changed again and the cells were additionally cultured for 24 h under arterial or venous pO2.
Ligand and Western blotting
Ligand blotting of conditioned media (CM) from primary hepatocytes using [125I]IGF-I as tracer was performed according to Hossenlopp et al. (31) with slight modifications as described recently (32, 33). Western blot analysis was carried out as described (30). The primary antibody against HIF-1, HIF-2, and HIF-3 were described earlier (27) and used in a 1:800 dilution. The primary monoclonal antibody against the V5-Tag was from Invitrogen and used in a 1:1000 dilution. Bound antibodies were detected by a goat antirabbit IgG-horseradish peroxidase (Santa Cruz Biotechnology, Heidelberg, Germany) or an antimouse IgG-horseradish peroxidase, respectively, used in a 1:2000 dilution. The ECL Western blotting system (Amersham Pharmacia Biotech, Braunschweig, Germany) was used for detection.
Isolation of total RNA and Northern blot analysis
Total RNA was isolated from rat liver cells as described recently (4, 34). For IGFBP-1, total RNA was evaluated using digoxigenin-labeled antisense RNAs generated by in vitro transcription with RNA polymerase (Roche, Mannheim, Germany) and the T3 promoter of pBluescript SK+ plasmid as described previously (33). Fragments of the rat IGFBP-4 plasmid (444 bp) were [32P]dCTP labeled by random priming. Prehybridization, hybridization, and washing of membranes were performed essentially as described (4, 34).
Statistical analysis
Autoradiographs of ligand and Northern blots were scanned (Gelcam Phase, Lübeck, Germany) and densitometrically analyzed (Molecular Analyst; Bio-Rad, Hercules, CA). The relative densities of bands were expressed as the percent increase or decrease compared with the respective untreated control and indicated as mean ± SD. The Student's t test for paired values with a P value of <0.05 was considered significant.
Results
Time course of O2-modulated expression of IGFBP-1 and IGFBP-4 mRNA and protein
To evaluate the effects of pO2 on IGFBP mRNA expression, rat hepatocytes were preincubated in serum-free medium for 1 h and then cultured in fresh serum-free medium under arterial (16% O2) or venous (8% O2) conditions for 6, 12, and 24 h. Under hypoxic conditions, IGFBP-1 mRNA abundance increased rapidly and reached maximal levels after 12 h of incubation (Fig. 1). In contrast, steady-state IGFBP-4 mRNA expression was stimulated under arterial pO2 with maximal levels after 12 h, whereas under venous pO2, IGFBP-4 mRNA remained at basal levels throughout the 24 h of culture (Fig. 1).
We evaluated the secretion of IGFBP proteins into CM of hepatocytes under arterial and venous conditions by [125I]IGF-I ligand blotting (Fig. 2) and Western blotting (data not shown). An increase in IGFBP-1 protein levels was detected after 6 h of culture under hypoxic conditions, and this effect persisted at all time points tested. After 12 h exposure to hypoxia, the amount of IGFBP-1 protein was increased 2-fold (Fig. 2). In contrast, maximal IGFBP-4 protein levels were detected under arterial pO2. After 24 h of culture, IGFBP-4 protein under venous pO2 was reduced by 35% compared with levels observed under arterial pO2 (Fig. 2).
To determine whether hypoxia regulates IGFBP-1 expression at the transcriptional level, rat hepatocytes were treated with the transcriptional inhibitor ActD (1 μg/ml) for 30 min and then placed under 16 or 8% pO2 for 6 and 12 h. Addition of ActD prevented the hypoxia-induced increase in IGFBP-1 mRNA at 6 h (Fig. 3) and reduced the secretion of IGFBP-1 protein after 12 h of cultivation under both arterial and venous conditions (Fig. 4) consistent with transcriptional regulation. ActD treatment of hepatocytes also prevented the normoxia-dependent induction of IGFBP-4 mRNA (Fig. 3) and IGFBP-4 protein (Fig. 4), although to a lesser extent than that observed for IGFBP-1.
Inhibition by H2O2 and enhancement by DSF of the venous pO2-dependent induction of IGFBP-1 mRNA
Iron- and redox-dependent processes have been shown to be involved in oxygen-regulated gene expression (35, 36, 37, 38, 39). We examined the effect of H2O2 (a generator of reactive oxygen species) on IGFBP-1 mRNA expression under arterial and venous pO2. H2O2 dose-dependently decreased IGFBP-1 mRNA levels under venous pO2, whereas IGFBP-1 mRNA expression under arterial pO2 was not affected by addition of H2O2 (Fig. 5).
The participation of iron in oxygen sensing was examined by treatment of rat hepatocytes with DSF, an iron chelator and inhibitor of heme synthesis with antioxidant properties. In the presence of 130 μM DSF, steady-state levels of IGFBP-1 mRNA were increased compared with the untreated control under both venous and arterial pO2 (Fig. 5).
Thus, H2O2 treatment simulated the effect of arterial pO2 and decreased the expression of IGFBP-1 mRNA under venous pO2, suggesting that reactive oxygen species may contribute to the effects of differences in oxygen tension on the expression of IGFBP-1. In contrast, chelation of iron caused by addition of DSF mimicked the effect of venous pO2 and further potentiated the induction of IGFBP-1 mRNA under both arterial and venous pO2, suggesting that iron may be important for O2-dependent IGFBP regulation.
HIF-3 contributes to the hypoxia-dependent induction of IGFBP-1 mRNA
HIFs have been shown to activate a number of genes under low O2 tension. To test whether HIFs activate IGFBP-1 expression in primary rat hepatocytes, we performed transient transfection studies with expression vectors for HIF-1, HIF-2, and HIF-3 and verified the expression of HIF-1, HIF-2, and HIF-3 by Western blotting (Fig. 6). Transfection experiments showed a HIF--dependent increase in IGFBP-1 mRNA expression under both venous and arterial pO2 when compared with the controls transfected with the empty expression vector. Interestingly, the most pronounced effects were observed in hepatocytes transfected with HIF-3, showing approximately 3-fold induction of IGFBP-1 mRNA expression under arterial pO2 and approximately 2-fold induction under venous pO2, respectively (Fig. 6). The expression of IGFBP-1 mRNA was increased by approximately 2-fold under arterial pO2 and approximately 1.5-fold under venous pO2 in cells transfected with the HIF-2 expression vector compared with the appropriate controls. Only a mild stimulation of IGFBP-1 mRNA expression was detectable after transfection with HIF-1 (Fig. 6). In contrast, transfection of hepatocytes with the different -subunits of HIF diminished the induction of IGFBP-4 mRNA under arterial pO2. Again, the stronger effects were found with HIF-3 and HIF-2 where transfection with HIF-2 and HIF-3 reduced the steady-state IGFBP-4 mRNA to about 65 and 45% of control levels under arterial pO2, whereas transfection with HIF-1 resulted only in a more modest decrease of IGFBP-4 mRNA abundance (Fig. 6). Similar alterations as observed at the IGFBP mRNA level after transfection with the different HIF vectors were also detected at the protein level (Fig. 7).
PHD-2 attenuates the hypoxia-dependent induction of IGFBP-1 mRNA
At normal oxygen tension, PHDs hydroxylate specific proline residues in the oxygen-dependent degradation domain of HIF -subunits to allow their binding to pVHL, which targets prolyl-hydroxylated HIF -subunits for degradation by the ubiquitin-proteasome system. To elucidate the role of PHDs in the regulation of hepatic IGFBP expression under normoxia and hypoxia, primary rat hepatocytes were transfected with expression vectors for PHD-1, PHD-2, PHD-3, and PHD-4 and subsequently cultivated under arterial and venous pO2. Expression of PHDs in hepatocytes was evaluated by Western blotting using anti-V5-Tag antibodies showing protein bands for PHD-1 and -2 at 44 and 46 kDa, respectively, for PHD-3 at 27 kDa and for PHD-4 at 56 and 50 kDa (Fig. 8). In addition, the effects of PHD transfections were also investigated on the level of the HIF -subunits. It was found that in PHD-2-transfected cells all three HIF -subunits were barely detectable, whereas PHD-3 was less effective but also decreasing the level of all three HIF -subunits, although a hypoxia-dependent induction was still apparent (Fig. 9). Transfection with PHD-1 and PHD-4 expression vectors only modestly reduced the level of HIF-1 under hypoxic conditions, whereas the effect of hypoxia on the expression of HIF-2 and HIF-3 was nearly abolished (Fig. 9).
Furthermore, transfection of hepatocytes with PHD-1, PHD-2, PHD-3, and PHD-4 consistently attenuated the induction of IGFBP-1 mRNA under venous conditions (Fig. 8). Although PHD-1 and PHD-4 transfection mediated only a slight decrease, PHD-2 and PHD-3 reduced IGFBP-1 mRNA levels to about 70 and 60%, respectively, of maximal levels observed in vector transfected hepatocytes under venous pO2.
Transfection with PHD vectors also altered the effect of arterial pO2 on the expression of IGFBP-4 mRNA. Transfection of PHD-1 decreased IGFBP-4 mRNA levels by approximately 30% under arterial pO2, whereas IGFBP-4 mRNA abundance was not significantly altered under venous conditions. Furthermore, PHD-2 reduced IGFBP-4 mRNA abundance by approximately 50% under arterial pO2 and about 20% under venous pO2. PHD-3 had an even more profound effect; decreasing IGFBP-4 mRNA levels by 70 and 85%, respectively. The transfection of PHD-4 had no effect on the IGFBP-4 mRNA levels (Fig. 8). The observed changes in IGFBP-1 mRNA levels after transfection with PHD vectors were reflected by changes in the levels of IGFBP proteins released into hepatocyte-CM (Fig. 9).
Together, these results indicate that IGFBP-1 and -4 expression in hepatocytes can be modulated via the HIF and PHD pathways with HIF-3 and PHD-2 playing a major role in this setting.
The sequence –252/–247 contributes to hypoxia-dependent induction of IGFBP-1
To test whether hypoxia exerts an effect on the IGFBP-1 promoter, we transfected an luciferase (Luc) reporter construct driven by the first 320 bp of the rat IGFBP-1 promoter. We found that venous conditions induced Luc activity by about 2-fold. Similarly, hypoxia mediated a 2-fold increase in Luc activity after transfection of a construct containing three HREs from the erythropoietin gene in front of the SV40 promoter. We next searched for the presence of the HRE consensus sequence within the rat IGFBP-1 promoter, and only the sequence 5'-CAAGTG-3' at –252/–247 resembled a 5'-RCGTG-3' HRE consensus sequence with one mismatch. Mutation of this sequence within the –320 IGFBP-1 promoter Luc construct abolished induction by hypoxic conditions. Likewise, mutation of the HIF-binding EPO-HRE also abolished induction by hypoxia (Fig. 10). Thus, the present data suggest that the sequence –252/–247 contributes to the hypoxia-dependent IGFBP-1 induction via binding of HIFs.
Discussion
The present study demonstrates that reduced oxygen tension enhances IGFBP-1 mRNA and protein expression in primary cultures of rat hepatocytes. In contrast, the expression of IGFBP-4 mRNA and protein are reduced by low pO2 conditions. The positive modulation of IGFBP-1 expression by venous pO2 was further stimulated by the iron chelator DSF, whereas H2O2 decreased IGFBP-1 mRNA levels, supporting the concept that iron and reactive oxygen species are involved in O2 sensing and the regulation of IGFBP-1. Transient transfection experiments demonstrated an involvement of HIF-3 and HIF-2 and to a lesser extent of HIF-1 for the induction of IGFBP-1 mRNA under venous conditions. The involvement of HIFs is further corroborated by experiments showing a role of PHDs in destabilization of HIFs, thereby attenuating the modulation by O2 of IGFBP-1 and -4 expression.
Oxygen as modulator of zonated gene expression
The results of the present study suggest that reduced pO2 in the perivenous region may constitute a critical determinant for the zonated expression of IGFBP-1 in rat liver. This is in line with findings indicating that hypoxia and the IGF system are interrelated (40). In vivo studies of rats fed with normal and high-protein diets showed higher levels of IGFBP-1 mRNA in the perivenous compared with the periportal zone of the liver (5). In addition, animal models of uteroplacental insufficiency (caused by uterine artery ligation or maternal hypoxia) resulted in fetal hypoxia and intrauterine growth restriction, with marked elevated circulating levels of IGFBP-1 and overexpression of IGFBP-1 mRNA in the fetal liver, the primary source of IGFBP-1 (40, 41, 42, 43). Furthermore, hypoxia stimulated IGFBP-1 expression in human HepG2 hepatoma cells as well as in primary cultures of human fetal hepatocytes (44, 45, 46).
Similar to IGFBP-1, the expression of glucokinase is greatest in the perivenous region of the hepatic acinus, and reduced pO2 stimulates the expression of glucokinase in isolated rat hepatocytes (47). In contrast, in this study we find that arterial pO2 is associated with increased expression of IGFBP-4 compared with low pO2. This is similar to previous results obtained with periportally expressed genes, including phosphoenolpyruvate carboxykinase (48, 49) and tyrosine aminotransferase (25).
In addition to the periportal to perivenous gradient of pO2 as a key regulator of the zonated gene expression, it also may be speculated that the gradient in IGFBP-1 expression is partly a result of changes in insulin levels across the liver acinus. Although up to 50% of insulin is degraded from the periportal to the perivenous zones (9), insulin receptor expression is higher in the perivenous zone (50), supporting the concept that the perivenous zone also is an important site of insulin action in the liver. By contrast, the glucagon receptor has been shown to be expressed predominantly in the periportal zone of the liver acinus (51). Because insulin is inhibitory (33) and glucagon is stimulatory (52, 53) to hepatic IGFBP-1 expression, the effects of insulin and glucagon are not likely to represent the underlying mechanism for the perivenous localization of IGFBP-1 expression in the liver.
Involvement of reactive oxygen species in O2 signaling
Reactive oxygen species derived from H2O2 have been proposed to be messengers of the O2 signal in hepatoma cells (36) as well as in hepatocytes (54). H2O2 is a highly diffusible molecule that is an ideal candidate for functioning as a second messenger that, in the presence of iron, yields highly reactive hydroxyl radicals via a local Fenton reaction (55). These radicals may lead to oxidation of proteins, thereby altering the function or stability of biologically active proteins, including transcription factors. H2O2 might mimic the effect of arterial pO2 by destabilizing the -subunit of the transcription factor HIF-1 and HIF-2 (56, 57). However, other studies report that HIF levels are enhanced in the presence of H2O2 (58, 59). In the present study, H2O2 decreased the induction of IGFBP-1 mRNA expression under venous pO2, thereby supporting the initial findings that H2O2 mimics the effect of arterial pO2, similar to its effects on the expression of PEPCK mRNA in primary hepatocytes (49, 55). Reciprocally, GK gene expression was down-regulated by H2O2 (47, 55). Thus, repression of hypoxia-dependent enhancement of IGFBP-1 mRNA by H2O2 further strengthens the proposal that H2O2 might function as a second messenger in regulation of IGFBP-1 expression by oxygen.
HIFs and HIF-prolyl hydroxylases
The oxygen-signaling system controlling induction of a variety of genes by hypoxia is present in most if not all cells and involves the induction of HIFs, which bind to HREs located in either the 5' or the 3' regions of the genes. Studies in human hepatoma cells have shown that HIF-1 can stimulate the expression of IGFBP-1 (44); however, the role of other HIFs such as HIF-2 or HIF-3 was not studied. The results of the present study suggest that the induction of IGFBP-1 by venous pO2 is mediated predominantly by HIF-3 and -2 and only to a lesser extent via HIF-1 in primary cultures of rat hepatocytes.
Under normoxia, HIF -subunit destabilization is mediated by O2-dependent hydroxylation of at least two proline residues within the oxygen-dependent degradation domain (13, 14, 15, 21, 60). The pVHL E3 ubiquitin ligase complex associates with hydroxylated proline residues and targets HIF -subunits for proteasomal degradation. Under hypoxia, oxygen becomes rate limiting for the at least four proline hydroxylase enzymes (PHDs) (15, 61). As a consequence, HIF -subunits accumulate, migrate into the nucleus, and associate with ARNT, and this complex interacts with HREs of target genes (60, 62). The results of the present study indicate a major role of PHD-2 in the destabilization of HIFs and reduced expression of IGFBP-1 under normoxic conditions in isolated hepatocytes. The effect of PHD-1, PHD-3, and PHD-4 was less prominent. These findings are largely consistent with another recent study showing that in Hela cells, PHD-2 was the major functional HIF-modifying enzyme, whereas PHD-1 and PHD-3 were ineffective, and PHD-4 was not investigated (63). The variation with respect to the action of PHD-3 may be eventually explained by cell-type-specific differences. Of note, because PHDs require Fe2+ in addition to O2, the induction of IGFBP-1 after treatment with DSF as observed in the present study might be a result of impairment of PHD activity, consistent with other studies (14, 15).
Interestingly, our study also showed that IGFBP-4 expression is repressed under hypoxia compared with normoxia. It might be speculated that higher gene expression under normoxia as shown in this study could be a result of an inhibitory effect of HIFs under hypoxia. The cotransfection experiments with the HIF expression vectors would support this because the normoxic IGFBP-4 levels are down-regulated after HIF -subunit transfection. Although HIFs act usually as activators, the role of HIFs as transcriptional inhibitors was also demonstrated in another study with the peroxisome proliferator-activated receptor- gene (64).
By contrast, the experiments using the PHD expression vectors do not completely support this concept because overexpression of PHD-1, -2, and -3 attenuated normoxic IGFBP-4 expression. This could be explained by the additional action of a so far unknown IGFBP-4 inducer that could be degraded by the action of PHDs.
Multiple regulatory elements have been identified in the IGFBP-1 promoter (65), including an insulin response element that mediates the negative effect of insulin on basal IGFBP-1 promoter activity and two glucocorticoid response elements. In addition, three consensus HREs have been identified in intron 1 of the human IGFBP-1 gene, including at least one that is hypoxia responsive with regard to IGFBP-1 gene expression. Recent studies suggest that other HREs located approximately 1000 bp upstream of the transcription initiation site may contribute to the regulation of IGFBP-1 in zebrafish and possibly other species (Kajimura, S., and C. Duan, personal communication). In the present study, we identified an HRE located approximately 250 bp upstream from the transcription initiation site in the rat IGFBP-1 promoter based on its relationship to the consensus HRE sequence. Functional studies confirmed that this element is necessary for the ability of hypoxia to stimulate IGFBP-1 promoter activity. This element is highly conserved in mouse and rat and partially conserved in the human IGFBP-1 promoter, and the ability of this site to bind and mediate effects of different HIFs on IGFBP-1 promoter activity is currently under investigation. Interestingly, this HRE resides in a region that has been reported to be important in enhancing effects of glucocorticoids on IGFBP-1 promoter activity (66), suggesting the interesting possibility that the interactions with this element may contribute cooperatively to the regulation of IGFBP-1 by other factors. Additional studies are required to unravel the role of this and other HREs in mediating the effects of hypoxia and other factors on IGFBP-1 expression. Additional studies regarding the mechanisms mediating the effects of high and low pO2 on the expression of IGFBP-4 also will be of significant interest.
In conclusion, the present study demonstrates that hypoxia enhances IGFBP-1 and reduces IGFBP-4 expression in primary rat hepatocytes. These studies provide additional evidence for the involvement of an oxygen-radical-generating process and the HIF system in oxygen sensing and in the regulation of zonated gene expression in the liver.
Acknowledgments
The technical assistance of Sieglinde Zachmann, Susanne Hupe, and Katja Curth is gratefully acknowledged.
Footnotes
This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 402, project A1 (to T.K.), GRK 335/2 (to T.K., J.-G.S.), and Scha 700/1-2 (to J.-G.S.); Georg-August-Universitt Gttingen, Schwerpunktfrderung Onkologie (to J.-G.S.), National Institutes of Health Grant DK41430 (to T.G.U.); and Department of Veterans Affairs Merit Review Program (to T.G.U.).
First Published Online September 15, 2005
Abbreviations: ActD, Actinomycin D; ARNT, aryl hydrocarbon receptor nuclear translocator; CM, conditioned medium; DSF, desferrioxamine; EPO, erythropoietin; HIF, hypoxia-inducible transcription factor; HRE, hypoxia-response element; IGFBP, IGF-binding protein; PHD, prolyl hydroxylase; pVHL, von Hippel-Lindau tumor suppressor protein.
Accepted for publication September 2, 2005.
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