Redox regulation of HIF-1alpha levels and HO-1 expression in renal medullary interstitial cells

Zhi-Zhang Yang, Andrew Y. Zhang, Fu-Xian Yi, Pin-Lan Li, and Ai-Ping Zou

Departments of Physiology and Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study hypothesized that superoxide (O<UP><SUB>2</SUB><SUP>−</SUP></UP>·) importantly contributes to the regulation of hypoxia-inducible factor (HIF)-1alpha expression at posttranscriptional levels in renal medullary interstitial cells (RMICs) of rats. By Western blot analysis, it was found that incubation of RMICs with O<UP><SUB>2</SUB><SUP>−</SUP></UP>· generators xanthine/xanthine oxidase and menadione significantly inhibited the hypoxia- or CoCl2-induced increase in HIF-1alpha levels and completely blocked the increase in HIF-1alpha levels induced by ubiquitin-proteasome inhibition with CBZ-LLL in the nuclear extracts from these cells. Under normoxic conditions, a cell-permeable O<UP><SUB>2</SUB><SUP>−</SUP></UP>· dismutase (SOD) mimetic, 4-hydroxyl-tetramethylpiperidin-oxyl (TEMPOL) and PEG-SOD, significantly increased HIF-1alpha levels in RMICs. Two mechanistically different inhibitors of NAD(P)H oxidase, diphenyleneiodonium and apocynin, were also found to increase HIF-1alpha levels in these renal cells. Moreover, introduction of an anti-sense oligodeoxynucleotide specific to NAD(P)H oxidase subunit, p22phox, into RMICs markedly increased HIF-1alpha levels. In contrast, the OH· scavenger tetramethylthiourea had no effect on the accumulation of HIF-1alpha in these renal cells. By Northern blot analysis, scavenging or dismutation of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· by TEMPOL and PEG-SOD was found to increase the mRNA levels of an HIF-1alpha -targeted gene, heme oxygenase-1. These results indicate that increased intracellular O<UP><SUB>2</SUB><SUP>−</SUP></UP>· levels induce HIF-1alpha degradation independently of H2O2 and OH· radicals in RMICs. NAD(P)H oxidase activity may importantly contribute to this posttranscriptional regulation of HIF-1alpha in these cells under physiological conditions.

reactive oxygen species; gene transcription; anoxia; renal interstitium; renal medulla


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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HYPOXIA-INDUCIBLE factor-1alpha (HIF-1alpha ) mediates the transcriptional activation of many oxygen-sensitive genes such as erythropoietin, heme oxygenase-1 (HO-1), inducible nitric oxide synthase, vascular endothelial growth factor, transferrin, and several glycolytic enzymes (18, 31-35, 37). This nuclear factor forms a heterodimer complex with its partner HIF-1beta to activate gene transcription. It has been demonstrated that HIF-1alpha can be induced by low tissue or cell O2 concentrations and rapidly degraded via an ubiquitin-proteasome pathway when O2 concentrations return to normoxic conditions (13, 16, 25). HIF-1beta constitutively appeared in cells and tissues under normoxic conditions. The HIF-1 heterodimer complex recognizes a DNA consensus sequence 5'-CGTG-3' in enhancer or promoter regions of many hypoxia-responsive genes, interacts with these binding sites in the major groove, and activates the transcription of these genes (32). Although much has been learned about the role of HIF-1 in activating the transcription of hypoxia-responsive genes, the mechanism by which HIF-1 levels within cells are regulated under physiological conditions is still poorly understood.

Reactive oxygen species (ROS) have been reported to be involved in the oxygen-sensing mechanism and play a critical role in the regulation of expression of oxygen-sensitive genes. Superoxide anions (O<UP><SUB>2</SUB><SUP>−</SUP></UP>·), hydrogen peroxide (H2O2), and hydroxyl radical (OH·) all have been implicated in the regulation of gene expression and related functions of different cells (17). Recent studies reported that changes in redox status mediate O2-dependent regulation of HIF-1alpha levels in different cell lines (2, 3, 12, 25), which may serve as an important mechanism activating oxygen-sensitive genes. However, it remains to be elucidated how ROS change HIF-1alpha levels and which species of ROS is importantly involved in the regulation of HIF-1alpha levels. More recently, we demonstrated that renal medullary cells more abundantly expressed HIF-1alpha compared with cortical cells and that HIF-1alpha participated in the transcriptional activation of oxygen-sensitive genes such as HO-1 (37, 46). The HIF-1alpha expression and related transcriptional activation of target genes in these cells may play an important role in the normal regulation of renal medullary oxygenation, renal medullary blood flow, and renal functions such as sodium excretion and osmolality adaptation (37, 43-46). However, the mechanism regulating HIF-1alpha levels in renal medullary cells is poorly understood. Given that ROS levels are higher in the renal medulla than cortex (44), it is possible that HIF-1alpha levels are importantly regulated by redox status in this kidney region. The present study used renal medullary interstitial cells (RMICs) as prototype cells to test whether HIF-1alpha levels in renal medullary cells are regulated by ROS and whether ROS may alter the transcriptional activation of some hypoxia-sensitive genes through a HIF-1alpha -mediated mechanism. Moreover, the present study examined which species of ROS contributes to the regulation of HIF-1alpha levels and whether NAD(P)H oxidase-derived O<UP><SUB>2</SUB><SUP>−</SUP></UP>· contributes to the redox regulation of HIF-1alpha expression in these kidney cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation and culture of RMICs. RMICs were isolated, cultured, and identified as we described previously (37). Briefly, inbred male Wistar rats weighing 300-350 g (Harlan Sprague Dawley, Madison, WI) were anesthetized with pentobarbital sodium (50 mg/kg body wt ip). Then, the left kidney was removed, and the renal papilla was dissected and finely minced. The minced tissue was resuspended in 3 ml of basic medium Eagle's (BME; Sigma) and injected subcutaneously in two to four vertical tracks on the abdominal wall of a recipient rat (from the same litter). Four days after injection, many firm and yellow nodules located at the site of injections were dissected carefully. These nodules were minced, trypsinized in 0.05% Trypsin-EDTA solution at 37°C for 20-30 min, and then washed and centrifuged to obtain a cell pellet. The cell suspension was transferred to plastic tissue culture flasks and then incubated with BME containing fetal bovine serum (10% vol/vol), amino acid mixtures (10% vol/vol), lactalbumin hydrolysate (0.25% wt/vol), yeast extracts (0.05% wt/vol), and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) using a 37°C incubator with a 95% air-5% CO2 environment. The culture medium was first replaced with fresh medium in 5 days and then changed every 3 days. These cells formed a confluent monolayer in 18-21 days and then were trypsinized and subsequently replanted in flasks. The cells from passages 7 and 8 were used for all experiments. The identity of these cells was confirmed by a standard staining method and light and electron microscopy as we described previously (37).

Preparation of nuclear extracts. Nuclear extracts from RMICs were prepared by a modification of the protocol described by Semenza and Wang (28). The cell pellet was washed with 4 packed-cell volumes (PCV) of buffer A [10 mM Tris · HCl (pH 7.8), 1.5 mM MgCl2, 10 mM KCl] containing 0.5 mM DTT, 0.4 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml pepstatin, 2 µg/ml aprotinin, and 1 mM sodium vanadate (all obtained from Sigma), resuspended in buffer A, and incubated on ice for 10 min. Then, the cell suspension was homogenized, and the nuclei were pelleted by centrifugation at 3,000 rpm for 5 min, resuspended in 3 PCV of buffer B [20 mM Tris · HCl (pH 7.8), 1.5 mM MgCl2, 0.42 M KCl, 20% glycerol] containing 0.5 mM DTT, 0.4 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml pepstatin, 2 µg/ml aprotinin, and 1 mM sodium vanadate, and mixed on a rotator at 4°C for 30 min. Finally, nuclear extracts were collected by centrifugation of nuclei incubation mixtures in buffer B for 30 min at 13,500 rpm. Aliquots were frozen in liquid N2 and stored at -80°C. Protein concentrations were determined using a Bio-Rad protein assay kit with bovine serum albumin standards. In our previous studies (37, 46), the nuclear extracts prepared according to this protocol were confirmed rich in HIF-1alpha .

Western blot analysis. Western blotting was performed as we described previously (37). Briefly, 40 µg of the nuclear extracts were subjected to 8% SDS-PAGE and transferred onto nitrocellulose membrane. Then, the membrane was washed and probed with 1:1,000 specific polyclonal anti-HIF-1alpha antibody and subsequently with 1:4,000 horseradish peroxidase-labeled goat anti-rabbit IgG. This polyclonal antibody against a 13-residue peptide from rat HIF-1alpha was prepared and validated in our previous studies (37, 46). To detect an immunoblotting signal, 10 ml of enhanced chemiluminescence detection solution (Amersham Pharmacia) were added, and the membrane was wrapped and exposed to Kodak Omat film. HIF-1beta was used as an internal control because HIF-1beta is constitutively expressed and not inducible during hypoxia, CoCl2, and other stimuli (13, 16, 25).

cDNA probes for Northern blot analysis of HIF-1alpha and HO-1. The HIF-1alpha and HO-1 cDNA from the rat kidney were cloned by RT-PCR with primer pairs designed and synthesized based on the sequences of rat HIF-1alpha and HO-1 cDNA in GenBank [accession number AF057308 for HIF-1alpha and M12129 for HO-1 (42, 46)]. A First-Strand cDNA Synthesis Kit (Amersham Pharmacia) was used to generate single-strand cDNA by RT, which was then used as a template for PCR with the primers for HIF-1alpha : 5'-CGGCGAAGCAAAGAGTCT-3' (sense) and 5'-TGAGGTTGGTTACTGTTG-3' (anti-sense); and for HO-1: 5'-GTCTATGCCCCGC TCTACTTC-3' (sense) and 5'-GTCTTAGCCTCTTCTGACACC-3' (anti-sense). The PCR products were fractionated on a 1.5% agarose gel, excised, and extracted with the use of a QIAGEN Gel Extraction Kit. The resulting cDNAs (542 bp for HIF-1alpha and 396 bp for HO-1) were cloned into pCR2.1-TOPO vector as described by the manufacturer (Invitrogen) and sequenced to confirm the identity of cDNA with an autosequencer by McConnell. The inserts for these genes in plasmid DNA were dissected by PCR or by enzyme digestion and used as probes for Northern blot analysis. The probes were purified and stored at -80°C until used.

RNA extraction and Northern blot analysis. Total RNA was extracted using TRIzol solution (Life Technologies) according to the manufacturer's protocol. Northern blot analyses of HIF-1alpha and HO-1 mRNAs were performed as described previously (37, 43, 46). In brief, total RNA (10-20 µg) was fractionated on a 1.0% formaldehyde-agarose gel, stained with ethidium bromide (0.5 µg/ml), washed, photographed, transferred onto nylon membrane (Pirece), and cross-linked to the membrane by UV irradiation. The nylon membranes were first prehybridized with Rapid Hyb buffer (Amersham Pharmacia) and then probed with 32P-labeled rat HIF-1alpha or HO-1 cDNA, respectively, at 65°C for 2.5 h. After being washed once at RT and then twice at 65°C, the membranes were autoradiographed at -80°C for 24 or 36 h. The autoradiographed films were scanned with a laser densitometer (Hewlett Packard ScanJet ADF) and then digitized by a UN-SCAN-IT software package (Silk Scientific). The densitometric values of those specific bands for corresponding gene expression were normalized to 28S rRNA.

Treatment of cells with various compounds. Xanthine and xanthine oxidase (X/XO; 100 µM · 50 mU-1 · ml-1) was used to produce O<UP><SUB>2</SUB><SUP>−</SUP></UP>· in the culture medium of RMICs, and menadione sodium bisulfite (100 µM) was used to stimulate production of endogenous O<UP><SUB>2</SUB><SUP>−</SUP></UP>· in these cells. The cell-permeable O<UP><SUB>2</SUB><SUP>−</SUP></UP>· scavengers 4-hydroxyl-tetramethylpiperidin-oxyl (TEMPOL; 0.1 mM) and PEG-SOD (50 U/ml) were used to decrease O<UP><SUB>2</SUB><SUP>−</SUP></UP>· levels in RMICs. Two mechanistically different NAD(P)H oxidase inhibitors, diphenyleneiodonium (DPI; 10 µM) and apocynin (100 µM), were used to inhibit O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production in RMICs. In additional groups of experiments, tetramethylthiourea (TMTU; 1 mM), an OH· scavenger, was used to remove OH· produced from H2O2 in the presence of TEMPOL and CBZ-LLL, a ubiquitin-proteasome inhibitor used to block HIF-1alpha degradation. All these reagents were directly added into the culture medium and incubated for a time period indicated in RESULTS. The doses or concentrations of these compounds for changing redox status in RMICs were chosen based on previous studies in our laboratory or by others, demonstrating that they effectively decreased or increased ROS levels in the cells or tissues (4, 11, 19, 21, 36, 37, 40, 44).

NAD(P)H oxidase subunit, p22phox anti-sense oligodeoxynucleotide transfection. p22phox Anti-sense oligodeoxynucleotide (P22-AS) was synthesized and introduced into RMICs as we previously described (37, 38). Briefly, a phosphorothioation-modified P22-AS was synthesized based on a cDNA sequence of p22phox (AJ295951) and it contained 5'-GCCCACTCGATCTGCCCCAT-3' (antisense, OPERON). The modification of five nucleotides on each side of P22-AS by phosphorothioation increased the stability and prevented this oligonucleotide from being degraded by intracellular nucleotide enzymes. The fluorescein attachment at the 5'-end was used as an indicator for transfection into the RMICs. The P22-AS was wrapped by cationic liposome (Avanti Polar Lipids, Alabaster, AL) and transfected into RMICs as described by the manufacturer. The transfection efficiency was evaluated by using a fluorescence microscope (Olympus, Tokyo, Japan) 3 h after incubation of RMICs with liposome-P22-AS mixtures. Positively transfected cells (70-80% cells) indicated by a remarkable intracellular fluorescence were used to determine the effects of p22phox blockade on HIF-1alpha levels.

Cell hypoxia. RMICs were plated in 100-mm2 tissue culture dishes 24 h before experiments and cultured to form a subconfluence. To decrease PO2 in the culture medium, these dishes were transferred to a sealed, humidified modular chamber and flushed for 2 h with 5% CO2-95% N2. PO2 in the culture medium was measured by a polarigraphic measurement described in our previous studies (45, 46), which is less than 10 mmHg after 2-h hypoxia.

Statistical analysis. Data are presented as means ± SE. The significance of difference in mean values within and between multiple groups was examined with an ANOVA for repeated measures followed by a Duncan's post hoc test. Student's t-test was used to evaluate the significance of differences between two groups of experiments (SigmaStat, SPSS). A value of P < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of X/XO and menadione on hypoxia- or CoCl2-induced HIF-1alpha protein expression in RMICs. Both X/XO and menadione have been commonly used to produce extracellular or intracellular O<UP><SUB>2</SUB><SUP>−</SUP></UP>·. X/XO, a O<UP><SUB>2</SUB><SUP>−</SUP></UP>·-generating enzyme system, significantly increased O<UP><SUB>2</SUB><SUP>−</SUP></UP>· levels in culture medium. O<UP><SUB>2</SUB><SUP>−</SUP></UP>· could enter into the cells at high concentrations. Menadione, a stimulator of mitochondrial O<UP><SUB>2</SUB><SUP>−</SUP></UP>·, increased the production of intracellular O<UP><SUB>2</SUB><SUP>−</SUP></UP>·. In our experiments, X/XO (100 µM · 50 mU-1 · ml-1) or menadione (100 µM) was added to cell culture medium and incubated for 22 h. Then, cells were subjected to hypoxia for another 2 h. It was found that HIF-1alpha protein levels significantly increased in control RMICs exposed to hypoxia for 2 h. However, the hypoxia-induced increase in HIF-1alpha protein levels was attenuated in RMICs pretreated with X/XO or menadione as shown in Fig. 1A. Similarly, HIF-1alpha protein levels were found to increase in RMICs treated by 150 µM CoCl2 for 4 h. In the presence of X/XO or menadione, the HIF-1alpha increase induced by CoCl2 was inhibited (Fig. 1B). All these experimental interventions had no effects on HIF-1beta levels. Summarized data from these experiments by densitometric analysis are presented in Fig. 1C. Increases in the intensity of the HIF-1alpha -immunoreactive band during hypoxia (n = 6) or treatment of CoCl2 (n = 6) were significantly attenuated by X/XO and menadione.


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Fig. 1.   Effects of xanthine/xanthine oxidase (X/XO) and menadione (MD) on hypoxia- or CoCl2-induced hypoxia-inducible factor (HIF)-1alpha protein expression in renal medullary interstitial cells (RMICs). A and B: typical ECL gel documents of Western blot analysis showing the HIF-1alpha protein levels in RMICs exposed to hypoxia for 2 h (H) or treated with 150 µM CoCl2 for 6 h in the presence or absence of X/XO (100 µM · 50 mU-1 · ml-1) or MD (100 µM). HIF-1beta was used as internal control in these experiments. C: summarized data showing the levels of HIF-1alpha protein in RMICs when treated by different stimuli. *P < 0.05, compared with control (C). #P < 0.05, compared with the values obtained during hypoxia or CoCl2 treatment alone.

Effects of X/XO and menadione on CBZ-LLL-induced increase in HIF-1alpha protein levels in RMICs. To further determine the effects of altered cell redox status on HIF-1alpha levels, we examined CBZ-LLL-induced alterations of HIF-1alpha levels in X/XO- or menadione-treated cells. CBZ-LLL is an inhibitor of ubiquitin-proteasome, which inhibits degradation of HIF-1alpha in the cells. In Fig. 2A are representative gel documents showing that treatment of RMICs with 10 µM CBZ-LLL for 4 h markedly increased HIF-1alpha levels. However, in the presence of X/XO or menadione, the CBZ-LLL-induced increase in HIF-1alpha protein levels was attenuated. In these experiments, HIF-1beta was not altered by any treatments. The data of these experiments are summarized in Fig. 2B. CBZ-LLL produced a 3.5-fold increase in HIF-1alpha protein levels in RMICs. In the presence of X/XO and menadione, the CBZ-LLL-induced increase in HIF-1alpha levels was completely blocked (n = 4).


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Fig. 2.   Effects of X/XO and MD on CBZ-LLL (CBZ)-induced HIF-1alpha protein levels in RMICs. A: typical ECL gel documents of HIF-1alpha protein expression in RMICs treated with 10 µM CBZ for 6 h in the presence or absence of X/XO or MD. B: summarized data showing HIF-1alpha protein levels during CBZ treatment in the absence or presence of X/XO or MD. *P < 0.05, compared with control. #P < 0.05, compared with the values obtained during CBZ treatment alone.

Effects of X/XO on HIF-1alpha mRNA expression in RMICs in response to hypoxia, CoCl2, and CBZ-LLL. To determine whether O<UP><SUB>2</SUB><SUP>−</SUP></UP>· affects HIF-1alpha mRNA expression, we detected changes in HIF-1alpha mRNA levels in RMICs under control conditions or subjected to different stimuli. It was found that HIF-1alpha mRNA levels increased in RMICs exposed to hypoxia for 2 h or to 150 µM CoCl2 for 4 h. Pretreatment of the cells with X/XO had no effect on hypoxia- and CoCl2-increased HIF-1alpha mRNA expression (Fig. 3A). Although CBZ-LLL significantly increased HIF-1alpha protein levels as shown above, it did not increase, even decreased and, HIF-1alpha mRNA expression by an unknown mechanism. However, in the presence of X/XO, this CBZ-LLL-decreased HIF-1alpha mRNA level was not further altered (Fig. 3B).


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Fig. 3.   Effects of X/XO on HIF-1alpha mRNA expression in RMICs in response to hypoxia, CoCl2, and CBZ. A: typical autoradiographic document of Northern blot analysis showing the mRNA levels of HIF-1alpha in RMICs exposed to hypoxia or treated with CoCl2 in the presence or absence of X/XO. B: typical autoradiographic document of Northern blot analysis showing the mRNA levels of HIF-1alpha in RMICs treated with CBZ in the presence or absence of X/XO.

Effects of TEMPOL, PEG-SOD, and DPI on HIF-1alpha levels in RMICs. To confirm the role of endogenously produced O<UP><SUB>2</SUB><SUP>−</SUP></UP>· in the regulation of HIF-1alpha levels, the effects of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· dismutation and production inhibition were examined. TEMPOL, a SOD mimetic, PEG-SOD, and DPI, a NAD(P)H oxidase inhibitor, have been widely used to decrease intracellular O<UP><SUB>2</SUB><SUP>−</SUP></UP>· levels (3, 4, 7). The present study used these compounds to reduce intracellular O<UP><SUB>2</SUB><SUP>−</SUP></UP>· levels and observed whether the decrease in intracellular O<UP><SUB>2</SUB><SUP>−</SUP></UP>· levels increased HIF-1alpha levels in RMICs. As shown in Fig. 4A, incubation of RMICs with TEMPOL (0.1 or 0.4 mM) or PEG-SOD (50 or 100 U/ml) for 6 h markedly increased HIF-1alpha protein levels in RMICs. Similarly, inhibition of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production by DPI (10 or 20 µM) for 6 h significantly increased HIF-1alpha protein levels in these cells. Figure 4B summarizes the results from these experiments. Both O<UP><SUB>2</SUB><SUP>−</SUP></UP>· dismutation and inhibition of its production significantly increased HIF-1alpha levels in RMICs (n = 6).


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Fig. 4.   Effects of endogenous O<UP><SUB>2</SUB><SUP>−</SUP></UP>· on HIF-1alpha protein levels in RMICs. A: representative gel documents showing the effects of treatment of RMICs with SOD mimetic TEMPOL (0.1 and 0.4 mM), PEG-SOD (50 and 100 U/ml), or NAD(P)H oxidase inhibitor DPI (10 and 20 µM) on HIF-1alpha levels. B: summarized data showing the HIF-1alpha protein levels in RMICs treated with TEMPOL, PEG-SOD, or DPI. *P < 0.05, compared with control.

Effects of apocynin and P22-AS on HIF-1alpha levels in RMICs. Although DPI has been reported to inhibit NAD(P)H oxidase and our recent study demonstrated that this compound at concentrations less than 50 µM had no effect on other O<UP><SUB>2</SUB><SUP>−</SUP></UP>·-generating enzyme systems such as XO and mitochondrial enzymes (38), as a flavoprotein oxidoreductase inhibitor the specificity of this compound to inhibit NAD(P)H oxidase activity has been often challenged. To address this issue, we performed two series of experiments to further determine the role of NAD(P)H oxidase-derived O<UP><SUB>2</SUB><SUP>−</SUP></UP>· in the regulation of HIF-1alpha levels in RMICs. In one group of experiments, apocynin, another specific inhibitor that blocks aggregation of NAD(P)H oxidase subunits and thereby inhibits its activity, was used (10, 29). As shown in the representative gel documents of Western blot analysis (Fig. 5A), apocynin (100 µM) markedly increased HIF-1alpha levels, but it was without effect on HIF-1beta expression. Figure 5B summarized the results from these experiments (n = 11), showing that HIF-1alpha , but not HIF-1beta levels were significantly increased in RMICs treated with apocynin.


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Fig. 5.   Effects of NAD(P)H oxidase inhibitor apocynin on HIF-1alpha protein levels in RMICs. A: representative gel documents showing the effects of treatment of RMICs with apocinin (100 µM) on HIF-1alpha and HIF-1beta levels. B: summarized data showing the HIF-1alpha and HIF-1beta protein levels in RMICs under control condition or treated with apocynin. *P < 0.05, compared with control.

In another group of experiments, we used an anti-sense oligodeoxynucleotide (ODN) approach to block the expression of NAD(P)H oxidase subunits and to examine the role of this enzyme in the regulation of HIF-1alpha levels in RMICs. Introduction of a P22-AS ODN into RMICs substantially blocked p22phox expression, as detected by Western blot analysis (data not shown). In these P22-AS ODN-transfected cells, HIF-1alpha levels were significantly increased compared with that in control cells or scrambled ODN-transfected cells. However, P22-AS had no effect on HIF-1beta levels in these RMICs (n = 11; Fig. 6).


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Fig. 6.   Effects of anti-sense oligodeoxynucelotides of p22phox (P22) on HIF-1alpha protein levels in RMICs. A: representative gel documents showing the effects of introduction of P22 on HIF-1alpha and HIF-1beta levels. B: summarized data showing the HIF-1alpha and HIF-1beta protein levels in RMICs under control condition or transfected with P22. *P < 0.05, compared with control.

Effects of TMTU on HIF-1alpha protein accumulation in RMICs in response to TEMPOL. Because H2O2 can be converted to form OH·, which was reported to increase the degradation of HIF-1alpha (12, 17, 25), we examined the effect of OH· production on HIF-1alpha expression associated with an H2O2 increase by TEMPOL. A specific scavenger of OH·, TMTU was used to examine the effects of OH· on HIF-1alpha protein levels in RMICs. Treatment of RMICs with 1 mM TMTU for 6 h had no effect on TEMPOL-induced increase in HIF-1alpha protein levels (n = 6; Fig. 7).


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Fig. 7.   Effects of tetramethylthiourea (TMTU) on HIF-1alpha protein accumulation in RMICs in response to TEMPOL. A: typical ECL gel document of Western blot analysis showing the HIF-1alpha protein levels in RMICs treated with hydroxyl radical (OH·) scavenger TMTU in response to TEMPOL. TMTU was used to study whether TEMPOL-induced HIF-1alpha increase in RMICs is related to OH· radical. B: summarized data showing the HIF-1alpha protein levels in RMICs treated with TMTU. *P < 0.05, compared with control.

Effects of TEMPOL and PEG-SOD on HO-1 mRNA expression in RMICs. Because the HO-1 gene is regulated transcriptionally by HIF-1alpha , we were wondering whether an endogenous O<UP><SUB>2</SUB><SUP>−</SUP></UP>·-induced decrease in HIF-1alpha levels influences the transcription of this gene. Therefore, one protocol was designed to examine the effects of TEMPOL and PEG-SOD on HO-1 mRNA expression. Consistent with an increase in HIF-1alpha protein levels, HO-1 mRNA expression in RMICs was significantly increased by TEMPOL and PEG-SOD (Fig. 8A). These results are summarized in Fig. 8B. Dismutation of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· by both TEMPOL (n = 4) and PEG-SOD (n = 4) significantly increased HO-1 mRNA expression.


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Fig. 8.   Effects of TEMPOL and PEG-SOD on HO-1 mRNA expression in RMICs. A: typical autoradiographic documents of Northern blot analysis showing the mRNA levels of HO-1 in RMICs treated with TEMPOL or PEG-SOD. B: summarized data showing the intensity ratio of HO-1 mRNA to 28S blots in RMICs under control condition and during treatments of TEMPOL or PEG-SOD. *P < 0.05, compared with control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It has been reported that the activation of many oxygen-sensitive genes during hypoxia is mediated by the binding of HIF-1, a HIF-1alpha and HIF-1beta complex, to a hypoxia-responsive element containing 5'-CGTG-3' in the promoter or enhancers of these genes (27, 28, 34, 35, 39). Because HIF-1alpha can respond to changes in PO2 at physiological range, this transcription factor has been considered as one of the most important transcription factors that are involved in the regulation of oxygen-sensitive genes (27, 34, 35). In previous studies, we demonstrated that HIF-1alpha mRNA and protein levels were enriched in the renal medulla, a kidney region exposed to low PO2 (less than 10 mmHg) under physiological conditions (46). RMICs isolated from this kidney region expressed HIF-1alpha even under normoxic conditions and in these cells HIF-1alpha could be increased in response to low PO2 or induced by different inducers such as desferrioxamine and CoCl2 (37). Therefore, RMICs represent an appropriate cell model to study the regulation of HIF-1alpha expression and to explore the functional significance of its regulatory mechanisms in the renal medulla.

In the first series of experiments, the exposure of these cells to hypoxia or HIF-1alpha inducer CoCl2 was found to significantly increase the levels of HIF-1alpha protein. In the presence of a continuous O<UP><SUB>2</SUB><SUP>−</SUP></UP>·-producing system X/XO, however, accumulation of HIF-1alpha protein during hypoxia or CoCl2 incubation was substantially blocked. This suggests that an increase in the production of ROS in RMICs downregulates HIF-1alpha levels, which may impair the adaptive response of many oxygen-sensitive genes to cell hypoxia. This inhibition of ROS on the response of HIF-1alpha to hypoxia in RMICs may be an important mechanism mediating the detrimental effects of ROS in this kidney region. To further determine whether intracellularly produced O<UP><SUB>2</SUB><SUP>−</SUP></UP>· contributes to the regulation of HIF-1alpha levels in RMICs, we examined the effects of an intracellular stimulator of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production, menadione, on HIF-1alpha protein levels and its response to hypoxia. It was found that menadione also significantly reduced hypoxia- or CoCl2-induced accumulation of HIF-1alpha protein in RMICs. These results demonstrate that intracellular O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production depresses HIF-1alpha increase in response to hypoxia, indicating that regardless of the resource of O<UP><SUB>2</SUB><SUP>−</SUP></UP>·, its increase in intracellular concentrations results in significant reduction of HIF-1alpha levels.

By Northern blot analysis, we found that X/XO did not have any significant effect on hypoxia- and CoCl2-induced upregulation of HIF-1alpha mRNA in RMICs. This suggests that the effect of ROS on HIF-1alpha levels may primarily occur at posttranscriptional levels, which is consistent with the previous studies indicating that ROS directly destabilize HIF-1alpha , resulting in its degradation through ubiquitin-proteasome (13, 16, 25). Indeed, treatment of RMICs with a selective ubiquitin-proteasome inhibitor, CBZ-LLL, significantly increased HIF-1alpha protein levels. In the presence of X/XO or menadione, however, the increase in HIF-1alpha protein levels induced by CBZ-LLL was substantially abolished. This antagonistic effect of ROS-generating systems on CBZ-LLL-induced inhibition of ubiquitin-proteasome indicates that ROS may enhance HIF-1alpha degradation associated with this proteasome system. This view is supported by a recent study showing that ROS directly activate a 26S ubiquitin-proteasome enzyme activity in K562 cells (24).

However, recent studies challenged this view regarding ROS-induced destabilization of HIF-1alpha . Especially, in nonhypoxic activation of this transcription factor, ROS seem to play a mediating role. For example, different cytokines or inflammatory factors such as TNF-alpha , IL-1beta , and thrombin have been reported to increase the mRNA or protein levels of HIF-1alpha or enhance its binding activity in several cell types, and inhibition of ROS production or increased ROS scavenging substantially blocked their effects on HIF-1alpha levels or activity (6, 8, 9). The reason for this discrepancy is still unclear. It is possible that there exist different regulatory mechanisms responsible for hypoxic and nonhypoxic activation of HIF-1alpha . Recent studies indicated that the different effects of ROS on HIF-1alpha levels or activity may be associated with the extent of oxidative stress. It has been proposed that moderate oxidative stress induces HIF-1alpha degradation by proteasomal system, whereas enhanced stress may inhibit the 26S proteasome, increasing HIF-1alpha levels or activity (23, 24, 26). On the basis of this view, moderate ROS production under normoxic conditions may decrease HIF-1alpha levels due to its degradation, but exaggerated production of ROS during inflammation or stimulation with inflammatory factors would increase HIF-1alpha levels or activity due to inhibition of proteasome. However, the present finding that incubation of the cells with X/XO and menadione largely decreased HIF-1alpha levels does not support this view, because it is obvious that exogenously induced oxidative stress by X/XO or menadione should not be a moderate oxidative stress in these cells. Considering a wide spectrum of the action of ROS on different signaling pathways, the exaggerated ROS production induced by X/XO or menadione may also alter HIF-1alpha levels or activity through other related regulatory pathways such as phosphorylation, cAMP signaling, and other mechanisms, which have been reported to regulate the activation or degradation of HIF-1alpha (8, 9, 26).

It is the diversity of exogenous ROS in stimulating or blocking HIF-1alpha production or degradation that prompted us to question the role of endogenously generated ROS in the regulation of HIF-1alpha levels in RMICs under normoxic conditions. The present study found that incubation of RMICs with either TEMPOL, a cell-permeable SOD mimetic, or PEG-SOD significantly increased HIF-1alpha protein concentrations. These results support the view that endogenously produced O<UP><SUB>2</SUB><SUP>−</SUP></UP>· anions exert a tonic regulatory action on HIF-1alpha levels in these renal cells, which maintains HIF-1alpha at appropriate intracellular levels. The results also suggest that endogenous H2O2 may not decrease HIF-1alpha levels in RMICs, because H2O2 produced by dismutation of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· with both PEG-SOD and TEMPOL did not exhibit the inhibitory effect on HIF-1alpha levels. It is O<UP><SUB>2</SUB><SUP>−</SUP></UP>· that decreases HIF-1alpha levels in RMICs. This conclusion is strengthened by the results obtained from the experiments using an OH· scavenger, which showed that scavenging OH· by TMTU had no effect on HIF-1alpha levels in RMICs irrespective of the absence or presence of SOD mimetic TEMPOL. However, these results are not consistent with those reported in previous studies in some cell lines such as Hela cells (17). In those studies, the generation of OH· from H2O2 via the iron-dependent Fenton reaction was proposed to stimulate the degradation of HIF-1alpha , thereby decreasing its levels in the cells. However, because those studies mainly examined the effects of exogenous H2O2 on HIF-1alpha protein levels, rather than that of endogenously produced H2O2, one should be cautious to conclude that H2O2 serves as an intracellular messenger molecule to mediate the redox response of HIF-1alpha as discussed above. Furthermore, recent studies demonstrated that H2O2 at high concentrations can produce O<UP><SUB>2</SUB><SUP>−</SUP></UP>· (1). It is possible that the effects of H2O2 on HIF-1alpha levels observed in those studies may simply be due to the production of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· when H2O2 is exogenously administrated at high concentrations. In addition, transformed cells used in those studies may behave differently in the redox regulation of HIF-1alpha compared with cultured normal RMICs. Taken together, our results suggest that the effect of endogenous O<UP><SUB>2</SUB><SUP>−</SUP></UP>· to decrease HIF-1alpha levels in RMICs is independent of H2O2 or OH·.

Next, we addressed whether NAD(P)H oxidase contributes to endogenous production of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· and thereby regulates HIF-1alpha levels in RMICs. NAD(P)H oxidase was chosen because this enzyme has been found to be a major enzyme to produce O<UP><SUB>2</SUB><SUP>−</SUP></UP>· in the renal medulla (44). It was found that HIF-1alpha protein levels increased in RMICs incubated with DPI, a NAD(P)H oxidase inhibitor that was used to characterize the activity of this enzyme pharmacologically (7), suggesting that NAD(P)H oxidase may be involved in the regulation of HIF-1alpha . As a flavoprotein oxidoreductase inhibitor, however, the specificity of DPI to inhibit NAD(P)H oxidase activity has been often challenged, despite that it was demonstrated to have no effect on other O<UP><SUB>2</SUB><SUP>−</SUP></UP>·-generating enzyme systems such as XO and mitochondrial enzymes at concentrations <50 µM, except NAD(P)H oxidase (38). With respect to HIF-1alpha , DPI was even found to block the stabilization of HIF-1alpha under certain circumstances (3, 5, 36). As discussed above, this opposite effect of DPI on HIF-1alpha levels may be associated with its use under conditions with a different extent of oxidative stress. However, the nonspecific effect may not be ruled out. To address this concern, we performed two additional series of experiments to confirm the role of NAD(P)H oxidase in the regulation of HIF-1alpha levels using a mechanistically different inhibitor, apocynin, and P22-AS. Consistently, both apocynin and P22-AS significantly increased HIF-1alpha levels in RMICs, but they had no effect on HIF-1beta levels. On the basis of these results, we believe that NAD(P)H oxidase as an endogenous resource of ROS may be importantly involved in the posttranscriptional regulation of HIF-1alpha in these renal medullary cells. In fact, recent studies also indicated that a nonphagocytic NAD(P)H oxidase [namely cytochrome b-type NAD(P)H oxidoreductase] may serve as an oxygen sensor to enhance degradation of HIF-1alpha through production of ROS (41, 42). It is possible that this NAD(P)H oxidase senses oxygen and produces O<UP><SUB>2</SUB><SUP>−</SUP></UP>·, which activates a proline hydroxylase in the presence of iron in the cytosol, resulting in hydroxylation of the proline residue in HIF-1alpha and ultimate degradation (41). Recently, this prolyl-4-hydroxylase-mediated hydroxylation of HIF-1alpha proline residue has been demonstrated to be necessary and sufficient for the binding of this transcription factor to von Hippel-Lindau protein, thereby resulting in its ubiquitination and degradation by the proteasome (14, 15, 20). However, the role of NAD(P)H oxidase in proline hydroxylation of HIF-1alpha remains to be determined.

The next question we tried to answer was whether O<UP><SUB>2</SUB><SUP>−</SUP></UP>·-mediated reduction of HIF-1alpha levels influences the transcriptional activation of those genes regulated by this transcription factor. In this regard, previous studies reported that ROS may increase HIF-1alpha degradation and thereby reduce the transcription of downstream genes (12, 17, 25). However, most of those studies were performed by exogenous addition of oxidants such as H2O2 and then detection of the changes in the expression of HIF-1alpha -targeted genes (12, 17, 22, 30). Little is known regarding the role of endogenously produced oxidants on the expression of those HIF-1alpha -targeted genes. With the use of HO-1 as a prototype gene, which was confirmed as a typical HIF-1alpha -activated gene (18, 37), the present study examined the effects of TEMPOL and PEG-SOD on HO-1 mRNA expression. Because TEMPOL and PEG-SOD have been found to increase HIF-1alpha levels as discussed above, it is expected that HO-1 mRNA levels should be increased. Indeed, we demonstrated that HO-1 mRNA expression was upregulated by TEMPOL and PEG-SOD. This suggests that endogenously produced O<UP><SUB>2</SUB><SUP>−</SUP></UP>· suppresses HO-1 gene expression, which is associated with decreases in HIF-1alpha in the cells.

In summary, the present studies provide evidence that HIF-1alpha levels in RMICs are regulated by cellular redox status in RMICs even under physiological conditions. NAD(P)H oxidase-derived O<UP><SUB>2</SUB><SUP>−</SUP></UP>· may represent one of the important resources of ROS to regulate HIF-1alpha levels. This redox regulation of HIF-1alpha may be one of the essential mechanisms maintaining normal levels of HIF-1alpha in renal medullary cells or resulting in tissue or cell injury during exaggerated oxidative stress in the kidney.


    ACKNOWLEDGEMENTS

This study was supported by National Institutes of Health Grants HL-70726 and DK-54927 and Grant 96007310 from the American Heart Association.


    FOOTNOTES

Address for reprint requests and other correspondence: A-P Zou, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226 (E-mail: azou{at}mcw.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 20, 2003;10.1152/ajprenal.00017.2002

Received 14 January 2002; accepted in final form 11 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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