Redox regulation of HIF-1 levels and HO-1 expression inrenal medullary intersti - 《American Journal of Physiology》美国生理学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 25 下一页

返回列表

查看: 526940\|回复: 247	go Redox regulation of HIF-1 levels and HO-1 expression inrenal medullary intersti [复制链接]

轻羽

新手上路

Rank: 1

积分: 0
威望: 0
包包: 0

楼主

发表于 2009-4-21 13:37 |只看该作者 |倒序浏览 |打印

作者：Zhi-ZhangYang, Andrew Y.Zhang, Fu-XianYi, Pin-LanLi,  Ai-PingZou作者单位：Departments of Physiology and Pharmacology andToxicology, Medical College of Wisconsin, Milwaukee, Wisconsin53226 3 c3 @  R' q. T0 [5 t3 ]
   6 A  D6 H* P$ [+ B) I, K
   8 ^( m$ ~0 }. D  b! h% N& Y
      0 v! N. m% u; G$ e9 a) a
                     ! t: w1 @/ c4 ?* E

         % m% U8 J! [- b/ M/ }$ Y  v( Q
         0 }9 v+ H9 ~. }: H( u7 c+ V# u$ c* e
         " r# T/ q1 }1 m0 f' `6 c


      ) E5 ]; @3 a+ p2 a  B

      【摘要】
   The present study hypothesized thatsuperoxide (O 2 − ·) importantly contributes to theregulation of hypoxia-inducible factor (HIF)-1 expression atposttranscriptional levels in renal medullary interstitial cells(RMICs) of rats. By Western blot analysis, it was found that incubationof RMICs with O 2 − · generators xanthine/xanthine oxidase and menadione significantly inhibited the hypoxia- or CoCl 2 -induced increase in HIF-1 levels and completelyblocked the increase in HIF-1 levels induced by ubiquitin-proteasome inhibition with CBZ-LLL in the nuclear extracts from these cells. Undernormoxic conditions, a cell-permeable O 2 − · dismutase(SOD) mimetic, 4-hydroxyl-tetramethylpiperidin-oxyl (TEMPOL) andPEG-SOD, significantly increased HIF-1 levels in RMICs. Twomechanistically different inhibitors of NAD(P)H oxidase, diphenyleneiodonium and apocynin, were also found to increase HIF-1 levels in these renal cells. Moreover, introduction of an anti-senseoligodeoxynucleotide specific to NAD(P)H oxidase subunit,p22 phox, into RMICs markedly increased HIF-1 levels. In contrast, the OH· scavenger tetramethylthiourea had noeffect on the accumulation of HIF-1 in these renal cells. ByNorthern blot analysis, scavenging or dismutation ofO 2 − · by TEMPOL and PEG-SOD was found to increase themRNA levels of an HIF-1 -targeted gene, heme oxygenase-1. Theseresults indicate that increased intracellular O 2 − ·levels induce HIF-1 degradation independently ofH 2 O 2 and OH· radicals in RMICs. NAD(P)H oxidase activity may importantly contribute to this posttranscriptional regulation of HIF-1 in these cells under physiological conditions. 2 ]7 _' f6 S, D2 T1 u2 _+ h7 h( S
      【关键词】 reactive oxygen species gene transcription anoxia renalinterstitium renal medulla2 F* [2 T) u( J' _7 _
   INTRODUCTION

HYPOXIA-INDUCIBLE factor-1 (HIF-1 ) mediates the transcriptional activation of manyoxygen-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 itspartner HIF-1 to activate gene transcription. It has beendemonstrated that HIF-1 can be induced by low tissue or cellO 2 concentrations and rapidly degraded via anubiquitin-proteasome pathway when O 2 concentrations returnto normoxic conditions ( 13, 16, 25 ). HIF-1 constitutively appeared in cells and tissues under normoxic conditions.The HIF-1 heterodimer complex recognizes a DNA consensus sequence5'-CGTG-3' in enhancer or promoter regions of many hypoxia-responsivegenes, interacts with these binding sites in the major groove, andactivates the transcription of these genes ( 32 ). Althoughmuch has been learned about the role of HIF-1 in activating thetranscription of hypoxia-responsive genes, the mechanism by which HIF-1levels within cells are regulated under physiological conditions isstill poorly understood.& n9 K: v; c) b

Reactive oxygen species (ROS) have been reported to be involved in theoxygen-sensing mechanism and play a critical role in the regulation ofexpression of oxygen-sensitive genes. Superoxide anions(O 2 − ·), hydrogen peroxide(H 2 O 2 ), and hydroxyl radical (OH·) all havebeen implicated in the regulation of gene expression and relatedfunctions of different cells ( 17 ). Recent studies reportedthat changes in redox status mediate O 2 -dependent regulation of HIF-1 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 ROSchange HIF-1 levels and which species of ROS is importantly involvedin the regulation of HIF-1 levels. More recently, we demonstratedthat renal medullary cells more abundantly expressed HIF-1 comparedwith cortical cells and that HIF-1 participated in thetranscriptional activation of oxygen-sensitive genes such as HO-1( 37, 46 ). The HIF-1 expression and relatedtranscriptional activation of target genes in these cells may play animportant 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-1 levels in renal medullarycells is poorly understood. Given that ROS levels are higher in therenal medulla than cortex ( 44 ), it is possible thatHIF-1 levels are importantly regulated by redox status in thiskidney region. The present study used renal medullary interstitialcells (RMICs) as prototype cells to test whether HIF-1 levels inrenal medullary cells are regulated by ROS and whether ROS may alterthe transcriptional activation of some hypoxia-sensitive genes througha HIF-1 -mediated mechanism. Moreover, the present study examinedwhich species of ROS contributes to the regulation of HIF-1 levelsand whether NAD(P)H oxidase-derived O 2 − · contributes to the redox regulation of HIF-1 expression in these kidney cells.
$ I6 m. q' g& ^6 l( w
MATERIALS AND METHODS6 ^" J% c  {+ T/ N' Z. K
! v5 \" n5 g, v
Isolation and culture of RMICs. RMICs were isolated, cultured, and identified as we describedpreviously ( 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 kidneywas removed, and the renal papilla was dissected and finely minced. Theminced tissue was resuspended in 3 ml of basic medium Eagle's (BME;Sigma) and injected subcutaneously in two to four vertical tracks onthe abdominal wall of a recipient rat (from the same litter). Four daysafter injection, many firm and yellow nodules located at the site ofinjections were dissected carefully. These nodules were minced,trypsinized in 0.05% Trypsin-EDTA solution at 37°C for 20-30min, and then washed and centrifuged to obtain a cell pellet. The cellsuspension was transferred to plastic tissue culture flasks and thenincubated with BME containing fetal bovine serum (10% vol/vol), aminoacid mixtures (10% vol/vol), lactalbumin hydrolysate (0.25% wt/vol),yeast extracts (0.05% wt/vol), and antibiotics (100 U/ml penicillinand 100 µg/ml streptomycin) using a 37°C incubator with a 95%air-5% CO 2 environment. The culture medium was firstreplaced with fresh medium in 5 days and then changed every 3 days.These cells formed a confluent monolayer in 18-21 days and thenwere 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 methodand light and electron microscopy as we described previously( 37 ).
! \* l2 R) x: M, M8 l2 P& R
Preparation of nuclear extracts. Nuclear extracts from RMICs were prepared by a modification of theprotocol described by Semenza and Wang ( 28 ). The cell pellet was washed with 4 packed-cell volumes (PCV) of bufferA [10 mM Tris · HCl (pH 7.8), 1.5 mMMgCl 2, 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 mMsodium vanadate (all obtained from Sigma), resuspended in bufferA, and incubated on ice for 10 min. Then, the cell suspension washomogenized, and the nuclei were pelleted by centrifugation at 3,000 rpm for 5 min, resuspended in 3 PCV of buffer B [20 mMTris · HCl (pH 7.8), 1.5 mM MgCl 2, 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 mMsodium vanadate, and mixed on a rotator at 4°C for 30 min. Finally,nuclear extracts were collected by centrifugation of nuclei incubationmixtures in buffer B for 30 min at 13,500 rpm. Aliquots werefrozen in liquid N 2 and stored at 80°C. Protein concentrations were determined using a Bio-Rad protein assay kit withbovine serum albumin standards. In our previous studies ( 37, 46 ), the nuclear extracts prepared according to this protocol were confirmed rich in HIF-1.
8 L( [4 S7 L/ P" _, }% ~
Western blot analysis. Western blotting was performed as we described previously( 37 ). Briefly, 40 µg of the nuclear extracts weresubjected to 8% SDS-PAGE and transferred onto nitrocellulose membrane.Then, the membrane was washed and probed with 1:1,000 specificpolyclonal anti-HIF-1 antibody and subsequently with 1:4,000horseradish peroxidase-labeled goat anti-rabbit IgG. This polyclonalantibody against a 13-residue peptide from rat HIF-1 was preparedand validated in our previous studies ( 37, 46 ). To detectan immunoblotting signal, 10 ml of enhanced chemiluminescence detectionsolution (Amersham Pharmacia) were added, and the membrane was wrapped and exposed to Kodak Omat film. HIF-1 was used as an internal control because HIF-1 is constitutively expressed and not inducible during hypoxia, CoCl 2, and other stimuli ( 13, 16, 25 ).. c  }; W" u4 d" u6 t

cDNA probes for Northern blot analysis of HIF-1 and HO-1. The HIF-1 and HO-1 cDNA from the rat kidney were cloned by RT-PCRwith primer pairs designed and synthesized based on the sequences ofrat HIF-1 and HO-1 cDNA in GenBank [accession number AF057308 forHIF-1 and M12129 for HO-1 ( 42, 46 )]. A First-StrandcDNA Synthesis Kit (Amersham Pharmacia) was used to generatesingle-strand cDNA by RT, which was then used as a template for PCRwith the primers for HIF-1 : 5'-CGGCGAAGCAAAGAGTCT-3' (sense) and5'-TGAGGTTGGTTACTGTTG-3' (anti-sense); and for HO-1: 5'-GTCTATGCCCCGCTCTACTTC-3' (sense) and 5'-GTCTTAGCCTCTTCTGACACC-3' (anti-sense). ThePCR products were fractionated on a 1.5% agarose gel, excised, andextracted with the use of a QIAGEN Gel Extraction Kit. The resultingcDNAs (542 bp for HIF-1 and 396 bp for HO-1) were cloned intopCR2.1-TOPO vector as described by the manufacturer (Invitrogen) andsequenced to confirm the identity of cDNA with an autosequencer byMcConnell. The inserts for these genes in plasmid DNA were dissected byPCR or by enzyme digestion and used as probes for Northern blotanalysis. The probes were purified and stored at 80°C until used.( l; e0 L* V5 M7 ~$ Y
- z1 }4 Q- M8 X* L+ m5 M- e
RNA extraction and Northern blot analysis. Total RNA was extracted using TRIzol solution (Life Technologies)according to the manufacturer's protocol. Northern blot analyses ofHIF-1 and HO-1 mRNAs were performed as described previously( 37, 43, 46 ). In brief, total RNA (10-20 µg) wasfractionated on a 1.0% formaldehyde-agarose gel, stained with ethidiumbromide (0.5 µg/ml), washed, photographed, transferred onto nylonmembrane (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 32 P-labeled ratHIF-1 or HO-1 cDNA, respectively, at 65°C for 2.5 h. Afterbeing washed once at RT and then twice at 65°C, the membranes wereautoradiographed at 80°C for 24 or 36 h. The autoradiographed films were scanned with a laser densitometer (Hewlett Packard ScanJetADF) and then digitized by a UN-SCAN-IT software package (SilkScientific). The densitometric values of those specific bands forcorresponding gene expression were normalized to 28S rRNA., `5 k$ {1 L. p$ a# r. X

Treatment of cells with various compounds. Xanthine and xanthine oxidase (X/XO; 100 µM · 50 mU 1 · ml 1 ) was usedto produce O 2 − · in the culture medium of RMICs, andmenadione sodium bisulfite (100 µM) was used to stimulate productionof endogenous O 2 − · in these cells. Thecell-permeable O 2 − · scavengers4-hydroxyl-tetramethylpiperidin-oxyl (TEMPOL; 0.1 mM) and PEG-SOD (50 U/ml) were used to decrease O 2 − · levels in RMICs.Two mechanistically different NAD(P)H oxidase inhibitors,diphenyleneiodonium (DPI; 10 µM) and apocynin (100 µM), were usedto inhibit O 2 − · production in RMICs. In additionalgroups of experiments, tetramethylthiourea (TMTU; 1 mM), an OH·scavenger, was used to remove OH· produced fromH 2 O 2 in the presence of TEMPOL and CBZ-LLL, aubiquitin-proteasome inhibitor used to block HIF-1 degradation. Allthese reagents were directly added into the culture medium andincubated for a time period indicated in RESULTS. The dosesor concentrations of these compounds for changing redox status in RMICswere chosen based on previous studies in our laboratory or by others,demonstrating that they effectively decreased or increased ROS levelsin the cells or tissues ( 4, 11, 19, 21, 36, 37, 40, 44 ).' T$ j9 ?1 i8 M3 V' U0 q# C9 S

NAD(P)H oxidase subunit, p22 phox anti-senseoligodeoxynucleotide transfection. p22 phox Anti-sense oligodeoxynucleotide (P22-AS)was synthesized and introduced into RMICs as we previously described( 37, 38 ). Briefly, a phosphorothioation-modifiedP22-AS was synthesized based on a cDNA sequence ofp22 phox ( AJ295951 ) and it contained5'-GCCCACTCGATCTGCCCCAT-3' (antisense, OPERON). The modification offive nucleotides on each side of P22-AS by phosphorothioation increasedthe stability and prevented this oligonucleotide from being degraded byintracellular nucleotide enzymes. The fluorescein attachment at the5'-end was used as an indicator for transfection into the RMICs. TheP22-AS was wrapped by cationic liposome (Avanti Polar Lipids,Alabaster, AL) and transfected into RMICs as described by themanufacturer. The transfection efficiency was evaluated by using afluorescence microscope (Olympus, Tokyo, Japan) 3 h afterincubation of RMICs with liposome-P22-AS mixtures. Positivelytransfected cells (70-80% cells) indicated by a remarkableintracellular fluorescence were used to determine the effects ofp22 phox blockade on HIF-1 levels.

Cell hypoxia. RMICs were plated in 100-mm 2 tissue culture dishes 24 h before experiments and cultured to form a subconfluence. To decrease P O 2 in the culture medium, these dishes weretransferred to a sealed, humidified modular chamber and flushed for2 h with 5% CO 2 -95% N 2.P O 2 in the culture medium was measured by apolarigraphic 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 differencein mean values within and between multiple groups was examined with anANOVA for repeated measures followed by a Duncan's post hoc test.Student's t -test was used to evaluate the significance ofdifferences between two groups of experiments (SigmaStat, SPSS). Avalue of P significant.
+ ~% |# Z( S( `. [$ e9 K
RESULTS

Effects of X/XO and menadione on hypoxia- orCoCl 2 -induced HIF-1 protein expression in RMICs. Both X/XO and menadione have been commonly used to produceextracellular or intracellular O 2 − ·. X/XO, aO 2 − ·-generating enzyme system, significantlyincreased O 2 − · levels in culture medium.O 2 − · could enter into the cells at highconcentrations. Menadione, a stimulator of mitochondrialO 2 − ·, increased the production of intracellularO 2 − ·. In our experiments, X/XO (100 µM · 50 mU 1 · ml 1 ) ormenadione (100 µM) was added to cell culture medium and incubated for22 h. Then, cells were subjected to hypoxia for another 2 h.It was found that HIF-1 protein levels significantly increased incontrol RMICs exposed to hypoxia for 2 h. However, thehypoxia-induced increase in HIF-1 protein levels was attenuated inRMICs pretreated with X/XO or menadione as shown in Fig. 1 A. Similarly, HIF-1 protein levels were found to increase in RMICs treated by 150 µMCoCl 2 for 4 h. In the presence of X/XO or menadione,the HIF-1 increase induced by CoCl 2 was inhibited (Fig. 1 B ). All these experimental interventions had no effects onHIF-1 levels. Summarized data from these experiments bydensitometric analysis are presented in Fig. 1 C. Increasesin the intensity of the HIF-1 -immunoreactive band during hypoxia( n = 6) or treatment of CoCl 2 ( n = 6) were significantly attenuated by X/XO andmenadione.

Fig. 1. Effects of xanthine/xanthine oxidase (X/XO) and menadione(MD) on hypoxia- or CoCl 2 -induced hypoxia-inducible factor(HIF)-1 protein expression in renal medullary interstitial cells(RMICs). A and B : typical ECL gel documents ofWestern blot analysis showing the HIF-1 protein levels in RMICsexposed to hypoxia for 2 h (H) or treated with 150 µMCoCl 2 for 6 h in the presence or absence of X/XO (100 µM · 50 mU 1 · ml 1 ) or MD (100 µM). HIF-1 was used as internal control in these experiments. C : summarized data showing the levels of HIF-1 protein inRMICs when treated by different stimuli. * P P 2 treatmentalone.
* J6 D. H* z  f* T2 I# N8 v. p) l
Effects of X/XO and menadione on CBZ-LLL-induced increase inHIF-1 protein levels in RMICs. To further determine the effects of altered cell redox status onHIF-1 levels, we examined CBZ-LLL-induced alterations of HIF-1 levels in X/XO- or menadione-treated cells. CBZ-LLL is an inhibitor ofubiquitin-proteasome, which inhibits degradation of HIF-1 in thecells. In Fig. 2 A arerepresentative gel documents showing that treatment of RMICs with 10 µM CBZ-LLL for 4 h markedly increased HIF-1 levels. However,in the presence of X/XO or menadione, the CBZ-LLL-induced increase inHIF-1 protein levels was attenuated. In these experiments, HIF-1 was not altered by any treatments. The data of these experiments aresummarized in Fig. 2 B. CBZ-LLL produced a 3.5-fold increasein HIF-1 protein levels in RMICs. In the presence of X/XO andmenadione, the CBZ-LLL-induced increase in HIF-1 levels wascompletely blocked ( n = 4).1 y, G$ D. d! L' D
9 `' P1 q" l) e# l  B5 h
Fig. 2. Effects of X/XO and MD on CBZ-LLL (CBZ)-inducedHIF-1 protein levels in RMICs. A : typical ECL geldocuments of HIF-1 protein expression in RMICs treated with 10 µMCBZ for 6 h in the presence or absence of X/XO or MD. B : summarized data showing HIF-1 protein levels duringCBZ treatment in the absence or presence of X/XO or MD.* P P

Effects of X/XO on HIF-1 mRNA expression in RMICs in response tohypoxia, CoCl 2, and CBZ-LLL. To determine whether O 2 − · affects HIF-1 mRNAexpression, we detected changes in HIF-1 mRNA levels in RMICs undercontrol conditions or subjected to different stimuli. It was found thatHIF-1 mRNA levels increased in RMICs exposed to hypoxia for 2 hor to 150 µM CoCl 2 for 4 h. Pretreatment of thecells with X/XO had no effect on hypoxia- andCoCl 2 -increased HIF-1 mRNA expression (Fig. 3 A ). Although CBZ-LLLsignificantly increased HIF-1 protein levels as shown above,it did not increase, even decreased and, HIF-1 mRNA expression by anunknown mechanism. However, in the presence of X/XO, thisCBZ-LLL-decreased HIF-1 mRNA level was not further altered (Fig. 3 B ).) ^: j8 P$ [) N3 k3 }4 x

Fig. 3. Effects of X/XO on HIF-1 mRNA expression in RMICs inresponse to hypoxia, CoCl 2, and CBZ. A : typicalautoradiographic document of Northern blot analysis showing the mRNAlevels of HIF-1 in RMICs exposed to hypoxia or treated withCoCl 2 in the presence or absence of X/XO. B :typical autoradiographic document of Northern blot analysis showing themRNA levels of HIF-1 in RMICs treated with CBZ in the presence orabsence of X/XO." X6 ^5 Y$ v$ n5 ~4 Y, \6 ^

Effects of TEMPOL, PEG-SOD, and DPI on HIF-1 levels inRMICs. To confirm the role of endogenously producedO 2 − · in the regulation of HIF-1 levels, theeffects of O 2 − · dismutation and productioninhibition were examined. TEMPOL, a SOD mimetic, PEG-SOD, and DPI, aNAD(P)H oxidase inhibitor, have been widely used to decreaseintracellular O 2 − · levels ( 3, 4, 7 ).The present study used these compounds to reduce intracellularO 2 − · levels and observed whether the decrease inintracellular O 2 − · levels increased HIF-1 levelsin RMICs. As shown in Fig. 4 A, 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-1 protein levels in RMICs.Similarly, inhibition of O 2 − · production by DPI (10 or 20 µM) for 6 h significantly increased HIF-1 proteinlevels in these cells. Figure 4 B summarizes the results fromthese experiments. Both O 2 − · dismutation andinhibition of its production significantly increased HIF-1 levels inRMICs ( n = 6).

Fig. 4. Effects of endogenous O 2 − · on HIF-1 protein levels in RMICs. A : representative gel documentsshowing 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 inhibitorDPI (10 and 20 µM) on HIF-1 levels. B : summarized datashowing the HIF-1 protein levels in RMICs treated with TEMPOL,PEG-SOD, or DPI. * P
; b% e& M: m% q3 T5 D( E
Effects of apocynin and P22-AS on HIF-1 levels in RMICs. Although DPI has been reported to inhibit NAD(P)H oxidase and ourrecent study demonstrated that this compound at concentrations lessthan 50 µM had no effect on other O 2 − ·-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 tofurther determine the role of NAD(P)H oxidase-derivedO 2 − · in the regulation of HIF-1 levels in RMICs.In one group of experiments, apocynin, another specific inhibitor thatblocks aggregation of NAD(P)H oxidase subunits and thereby inhibits itsactivity, was used ( 10, 29 ). As shown in therepresentative gel documents of Western blot analysis (Fig. 5 A ), apocynin (100 µM)markedly increased HIF-1 levels, but it was without effect onHIF-1 expression. Figure 5 B summarized the results fromthese experiments ( n = 11), showing that HIF-1, butnot HIF-1 levels were significantly increased in RMICs treated withapocynin.
2 q9 Q1 V7 H6 O5 ^! y
Fig. 5. Effects of NAD(P)H oxidase inhibitor apocynin on HIF-1 protein levels in RMICs. A : representative gel documentsshowing the effects of treatment of RMICs with apocinin (100 µM) onHIF-1 and HIF-1 levels. B : summarized data showing theHIF-1 and HIF-1 protein levels in RMICs under control conditionor treated with apocynin. * P% q9 _" T; D/ A% f
* Q! V6 {5 i' p$ N5 ^
In another group of experiments, we used an anti-senseoligodeoxynucleotide (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-1 levels in RMICs. Introduction of a P22-AS ODNinto RMICs substantially blocked p22 phox expression, as detected by Western blot analysis (data not shown). Inthese P22-AS ODN-transfected cells, HIF-1 levels were significantly increased compared with that in control cells or scrambledODN-transfected cells. However, P22-AS had no effect on HIF-1 levelsin these RMICs ( n = 11; Fig. 6 ).2 I/ p* u* o6 m7 w

Fig. 6. Effects of anti-sense oligodeoxynucelotides ofp22 phox (P22) on HIF-1 protein levels inRMICs. A : representative gel documents showing the effectsof introduction of P22 on HIF-1 and HIF-1 levels. B :summarized data showing the HIF-1 and HIF-1 protein levels inRMICs under control condition or transfected with P22.* P

Effects of TMTU on HIF-1 protein accumulation in RMICs inresponse to TEMPOL. Because H 2 O 2 can be converted to form OH·,which was reported to increase the degradation of HIF-1 ( 12, 17, 25 ), we examined the effect of OH· production on HIF-1 expression associated with an H 2 O 2 increase byTEMPOL. A specific scavenger of OH·, TMTU was used to examine theeffects of OH· on HIF-1 protein levels in RMICs. Treatment ofRMICs with 1 mM TMTU for 6 h had no effect on TEMPOL-inducedincrease in HIF-1 protein levels ( n = 6; Fig. 7 ).
4 O$ f4 ~4 I. h9 }
Fig. 7. Effects of tetramethylthiourea (TMTU) on HIF-1 proteinaccumulation in RMICs in response to TEMPOL. A : typical ECLgel document of Western blot analysis showing the HIF-1 proteinlevels in RMICs treated with hydroxyl radical (OH·) scavenger TMTU inresponse to TEMPOL. TMTU was used to study whether TEMPOL-inducedHIF-1 increase in RMICs is related to OH· radical. B :summarized data showing the HIF-1 protein levels in RMICs treatedwith TMTU. * P

Effects of TEMPOL and PEG-SOD on HO-1 mRNA expression in RMICs. Because the HO-1 gene is regulated transcriptionally by HIF-1, wewere wondering whether an endogenous O 2 − ·-induced decrease in HIF-1 levels influences the transcription of this gene.Therefore, one protocol was designed to examine the effects of TEMPOLand PEG-SOD on HO-1 mRNA expression. Consistent with an increase inHIF-1 protein levels, HO-1 mRNA expression in RMICs wassignificantly increased by TEMPOL and PEG-SOD (Fig. 8 A ). These results aresummarized in Fig. 8 B. Dismutation ofO 2 − · by both TEMPOL ( n = 4) andPEG-SOD ( n = 4) significantly increased HO-1 mRNAexpression.( R* m' r+ }5 D0 k/ ~) `/ P% O' D3 M
. s: [& v. M% J* \
Fig. 8. Effects of TEMPOL and PEG-SOD on HO-1 mRNA expression inRMICs. A : typical autoradiographic documents of Northernblot analysis showing the mRNA levels of HO-1 in RMICs treated withTEMPOL or PEG-SOD. B : summarized data showing the intensityratio of HO-1 mRNA to 28S blots in RMICs under control condition andduring treatments of TEMPOL or PEG-SOD. * P
# l/ s+ K6 O1 }) `: K
DISCUSSION! H0 N" x9 \  W1 a0 }$ b! r
+ |1 r  N2 g+ e0 y( `9 O1 d/ f
It has been reported that the activation of many oxygen-sensitivegenes during hypoxia is mediated by the binding of HIF-1, a HIF-1 and HIF-1 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-1 can respond to changes inP O 2 at physiological range, this transcriptionfactor has been considered as one of the most important transcriptionfactors that are involved in the regulation of oxygen-sensitive genes ( 27, 34, 35 ). In previous studies, we demonstrated that HIF-1 mRNA and protein levels were enriched in the renal medulla, akidney region exposed to low P O 2 (less than 10 mmHg) under physiological conditions ( 46 ). RMICs isolatedfrom this kidney region expressed HIF-1 even under normoxicconditions and in these cells HIF-1 could be increased in responseto low P O 2 or induced by different inducerssuch as desferrioxamine and CoCl 2 ( 37 ).Therefore, RMICs represent an appropriate cell model to study theregulation of HIF-1 expression and to explore the functionalsignificance of its regulatory mechanisms in the renal medulla.

In the first series of experiments, the exposure of these cells tohypoxia or HIF-1 inducer CoCl 2 was found tosignificantly increase the levels of HIF-1 protein. In the presenceof a continuous O 2 − ·-producing system X/XO, however, accumulation of HIF-1 protein during hypoxia or CoCl 2 incubation was substantially blocked. This suggests that an increase inthe production of ROS in RMICs downregulates HIF-1 levels, which mayimpair the adaptive response of many oxygen-sensitive genes to cellhypoxia. This inhibition of ROS on the response of HIF-1 to hypoxiain RMICs may be an important mechanism mediating the detrimentaleffects of ROS in this kidney region. To further determine whetherintracellularly produced O 2 − · contributes to theregulation of HIF-1 levels in RMICs, we examined the effects of anintracellular stimulator of O 2 − · production,menadione, on HIF-1 protein levels and its response to hypoxia. Itwas found that menadione also significantly reduced hypoxia- orCoCl 2 -induced accumulation of HIF-1 protein in RMICs. These results demonstrate that intracellular O 2 − · production depresses HIF-1 increase in response to hypoxia,indicating that regardless of the resource of O 2 − ·, its increase in intracellular concentrations results in significant reduction of HIF-1 levels.2 |7 A' U- X5 p# E5 P% H  o

By Northern blot analysis, we found that X/XO did not have anysignificant effect on hypoxia- and CoCl 2 -inducedupregulation of HIF-1 mRNA in RMICs. This suggests that the effectof ROS on HIF-1 levels may primarily occur at posttranscriptionallevels, which is consistent with the previous studies indicating that ROS directly destabilize HIF-1, resulting in its degradation throughubiquitin-proteasome ( 13, 16, 25 ). Indeed, treatment ofRMICs with a selective ubiquitin-proteasome inhibitor, CBZ-LLL, significantly increased HIF-1 protein levels. In the presence ofX/XO or menadione, however, the increase in HIF-1 protein levelsinduced by CBZ-LLL was substantially abolished. This antagonistic effect of ROS-generating systems on CBZ-LLL-induced inhibition ofubiquitin-proteasome indicates that ROS may enhance HIF-1 degradation associated with this proteasome system. This view issupported by a recent study showing that ROS directly activate a 26Subiquitin-proteasome enzyme activity in K562 cells ( 24 ).

However, recent studies challenged this view regarding ROS-induceddestabilization of HIF-1. Especially, in nonhypoxic activation ofthis transcription factor, ROS seem to play a mediating role. Forexample, different cytokines or inflammatory factors such as TNF-,IL-1, and thrombin have been reported to increase the mRNA orprotein levels of HIF-1 or enhance its binding activity in severalcell types, and inhibition of ROS production or increased ROSscavenging substantially blocked their effects on HIF-1 levels oractivity ( 6, 8, 9 ). The reason for this discrepancy isstill unclear. It is possible that there exist different regulatory mechanisms responsible for hypoxic and nonhypoxic activation of HIF-1. Recent studies indicated that the different effects of ROS onHIF-1 levels or activity may be associated with the extent ofoxidative stress. It has been proposed that moderate oxidative stress induces HIF-1 degradation by proteasomal system, whereas enhanced stress may inhibit the 26S proteasome, increasing HIF-1 levels or activity ( 23, 24, 26 ). On the basis of thisview, moderate ROS production under normoxic conditions may decrease HIF-1 levels due to its degradation, but exaggerated production ofROS during inflammation or stimulation with inflammatory factors wouldincrease HIF-1 levels or activity due to inhibition of proteasome.However, the present finding that incubation of the cells with X/XO andmenadione largely decreased HIF-1 levels does not support this view,because it is obvious that exogenously induced oxidative stress by X/XOor menadione should not be a moderate oxidative stress in these cells.Considering a wide spectrum of the action of ROS on different signalingpathways, the exaggerated ROS production induced by X/XO or menadionemay also alter HIF-1 levels or activity through other relatedregulatory pathways such as phosphorylation, cAMP signaling, and othermechanisms, which have been reported to regulate the activation ordegradation of HIF-1 ( 8, 9, 26 ).
) ]0 J1 N$ Z8 Z- m5 B9 k+ X
It is the diversity of exogenous ROS in stimulating or blockingHIF-1 production or degradation that prompted us to question therole of endogenously generated ROS in the regulation of HIF-1 levelsin RMICs under normoxic conditions. The present study found thatincubation of RMICs with either TEMPOL, a cell-permeable SOD mimetic,or PEG-SOD significantly increased HIF-1 protein concentrations.These results support the view that endogenously producedO 2 − · anions exert a tonic regulatory action onHIF-1 levels in these renal cells, which maintains HIF-1 atappropriate intracellular levels. The results also suggest thatendogenous H 2 O 2 may not decrease HIF-1 levels in RMICs, because H 2 O 2 produced bydismutation of O 2 − · with both PEG-SOD and TEMPOL didnot exhibit the inhibitory effect on HIF-1 levels. It isO 2 − · that decreases HIF-1 levels in RMICs. Thisconclusion is strengthened by the results obtained from the experimentsusing an OH· scavenger, which showed that scavenging OH· by TMTUhad no effect on HIF-1 levels in RMICs irrespective of the absenceor presence of SOD mimetic TEMPOL. However, these results are notconsistent with those reported in previous studies in some cell linessuch as Hela cells ( 17 ). In those studies, the generationof OH· from H 2 O 2 via the iron-dependentFenton reaction was proposed to stimulate the degradation of HIF-1,thereby decreasing its levels in the cells. However, because thosestudies mainly examined the effects of exogenousH 2 O 2 on HIF-1 protein levels, rather thanthat of endogenously produced H 2 O 2, one shouldbe cautious to conclude that H 2 O 2 serves as anintracellular messenger molecule to mediate the redox response ofHIF-1 as discussed above. Furthermore, recent studies demonstratedthat H 2 O 2 at high concentrations can produceO 2 − · ( 1 ). It is possible that theeffects of H 2 O 2 on HIF-1 levels observed inthose studies may simply be due to the production ofO 2 − · when H 2 O 2 isexogenously administrated at high concentrations. In addition,transformed cells used in those studies may behave differently in theredox regulation of HIF-1 compared with cultured normal RMICs. Takentogether, our results suggest that the effect of endogenousO 2 − · to decrease HIF-1 levels in RMICs isindependent of H 2 O 2 or OH·.

Next, we addressed whether NAD(P)H oxidase contributes to endogenousproduction of O 2 − · and thereby regulates HIF-1 levels in RMICs. NAD(P)H oxidase was chosen because this enzyme hasbeen found to be a major enzyme to produce O 2 − · inthe renal medulla ( 44 ). It was found that HIF-1 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-1. As a flavoproteinoxidoreductase inhibitor, however, the specificity of DPI to inhibitNAD(P)H oxidase activity has been often challenged, despite that it was demonstrated to have no effect on otherO 2 − ·-generating enzyme systems such as XO andmitochondrial enzymes at concentrations 38 ). With respect to HIF-1, DPI was even foundto block the stabilization of HIF-1 under certain circumstances( 3, 5, 36 ). As discussed above, this opposite effect ofDPI on HIF-1 levels may be associated with its use under conditionswith a different extent of oxidative stress. However, the nonspecificeffect may not be ruled out. To address this concern, we performed twoadditional series of experiments to confirm the role of NAD(P)H oxidasein the regulation of HIF-1 levels using a mechanistically differentinhibitor, apocynin, and P22-AS. Consistently, both apocynin and P22-ASsignificantly increased HIF-1 levels in RMICs, but they had noeffect on HIF-1 levels. On the basis of these results, we believethat NAD(P)H oxidase as an endogenous resource of ROS may beimportantly involved in the posttranscriptional regulation of HIF-1 in these renal medullary cells. In fact, recent studies also indicatedthat a nonphagocytic NAD(P)H oxidase [namely cytochrome b -type NAD(P)H oxidoreductase] may serve as an oxygensensor to enhance degradation of HIF-1 through production of ROS( 41, 42 ). It is possible that this NAD(P)H oxidase sensesoxygen and produces O 2 − ·, which activates a prolinehydroxylase in the presence of iron in the cytosol, resulting inhydroxylation of the proline residue in HIF-1 and ultimatedegradation ( 41 ). Recently, thisprolyl-4-hydroxylase-mediated hydroxylation of HIF-1 proline residuehas been demonstrated to be necessary and sufficient for the binding ofthis transcription factor to von Hippel-Lindau protein, therebyresulting in its ubiquitination and degradation by the proteasome( 14, 15, 20 ). However, the role of NAD(P)H oxidase inproline hydroxylation of HIF-1 remains to be determined.% D* [- Y) J* }- w: {5 C
4 \# p9 x1 B1 \5 K" ]: A4 e" c, w
The next question we tried to answer was whetherO 2 − ·-mediated reduction of HIF-1 levelsinfluences the transcriptional activation of those genes regulated bythis transcription factor. In this regard, previous studies reported that ROS may increase HIF-1 degradation and thereby reduce the transcription of downstream genes ( 12, 17, 25 ). However, most of those studies were performed by exogenous addition of oxidantssuch as H 2 O 2 and then detection of the changesin the expression of HIF-1 -targeted genes ( 12, 17, 22, 30 ). Little is known regarding the role of endogenously producedoxidants on the expression of those HIF-1 -targeted genes. With theuse of HO-1 as a prototype gene, which was confirmed as a typical HIF-1 -activated gene ( 18, 37 ), the present studyexamined the effects of TEMPOL and PEG-SOD on HO-1 mRNA expression.Because TEMPOL and PEG-SOD have been found to increase HIF-1 levelsas 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 2 − · suppresses HO-1 gene expression, whichis associated with decreases in HIF-1 in the cells.
) s) D1 u! ?2 Z: z
In summary, the present studies provide evidence that HIF-1 levelsin RMICs are regulated by cellular redox status in RMICs even underphysiological conditions. NAD(P)H oxidase-derivedO 2 − · may represent one of the important resources ofROS to regulate HIF-1 levels. This redox regulation of HIF-1 maybe one of the essential mechanisms maintaining normal levels ofHIF-1 in renal medullary cells or resulting in tissue or cell injuryduring exaggerated oxidative stress in the kidney.. N3 i  y% K) a
' W$ R/ w. ^( o( f" `2 \
ACKNOWLEDGEMENTS
) i" k. M* P; |! j+ W
This study was supported by National Institutes of Health GrantsHL-70726 and DK-54927 and Grant 96007310 from the American Heart Association.
      【参考文献】8 D( c/ d: j7 u, k
1. Carter, WO,Narayanan PK,andRobinson JP. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J Leukoc Biol 55:253-258,1994.
' U4 A) A; G/ j0 ]$ p

* R- q6 i6 `- y& d7 b
2. Chandel, NS,Maltepe E,Goldwasser E,Mathieu CE,Simon MC,andSchumacker PT. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci USA 95:11715-11720,1998.  ~9 C* C3 F/ F/ a1 J
6 D7 o, e: Z5 }$ E  j! c
5 L% a( a% z8 g7 z
- n1 X# a# `& d- p. N9 [
3. Chandel, NS,McClintock DS,Feliciano CE,Wood TM,Melendez JA,Rodriguez AM,andSchumacker PT. Reactive oxygen species generated at mitochondrial complex I.I.I. stabilize hypoxia-inducible factor-1 during hypoxia: a mechanism of O 2 sensing. J Biol Chem 275:25130-25138,2000.) e$ J: {6 S8 q' ]& Y& [

7 A9 d7 @1 J+ n" y$ P" }
4. Chen, YF,Li PL,andZou AP. Oxidative stress enhances the production and actions of adenosine in the kidney. Am J Physiol Regul Integr Comp Physiol 281:R1808-R1816,2001.* k; v) a  O9 F- A" _

! t2 n$ k$ q7 S4 g. A

5. Gleadle, JM,Ebert BL,andRatcliffe PJ. Diphenylene iodonium inhibits the induction of erythropoietin and other mammalian genes by hypoxia. Implications for the mechanism of oxygen sensing. Eur J Biochem 234:92-99,1995.
/ b: z: T! @" o% l! U
0 T+ k7 S# J  U
% y& r3 x# \6 W
6. Gorlach, A,Diebold I,Schini-Kerth VB,Berchner-Pfannschmidt U,Roth U,Brandes RP,Kietzmann T,andBusse R. Thrombin activates the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells: role of the p22 phox -containing NADPH oxidase. Circ Res 89:47-54,2001.% I* {: S0 y2 h' r

4 r/ H$ t# [, y8 X( K# V4 Z6 ]; \+ Q
7. Griendling, KK,Sorescu D,andUshio-Fukai M. NAD(P)H cxidase: role in cardiovascular biology and disease. Circ Res 86:494-501,2000., n' c! B* M3 O
! i+ M) F$ ]0 T2 e; w" R+ o
9 c! h' i, h' k3 F" F$ N& I3 B2 |

8. Haddad, JJ. Recombinant human interleukin (IL)-1 -mediated regulation of hypoxia-inducible factor-1 (HIF-1 ) stabilization, nuclear translocation and activation requires an antioxidant/reactive oxygen species (ROS)-sensitive mechanism. Eur Cytokine Netw 13:250-260,2002.+ J5 P( n* _% ]% W

. r1 l7 R8 \; X% Y8 f

9. Haddad, JJ,andLand SC. A nonhypoxic, ROS-sensitive pathway mediates TNF- -dependent regulation of HIF-1. FEBS Lett 505:269-274,2001.6 J2 r, c' e: V: O( l* I

- T( T5 w' |  Q  H3 t0 h

10. Hamilton, CA,Brosnan MJ,Al-Benna S,Berg G,andDominiczak AF. NAD(P)H oxidase inhibition improves endothelial function in rat and human blood vessels. Hypertension 40:755-762,2002.

4 l8 T& S% N8 N
11. Heberlein, W,Wodopia R,Bartsch P,andMairbaurl H. Possible role of ROS as mediators of hypoxia-induced ion transport inhibition of alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 278

640-L648,2000.  D" `2 i) h, u9 ?3 F
1 ?  P6 E2 t! ~7 `
1 m) c% V! R7 o* T, ~9 {3 s6 k
; V2 a. E9 ?* T/ t/ q' w0 T
12. Huang, LE,Arany Z,Livingston DM,andBunn HF. Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit. J Biol Chem 271:32253-32259,1996.% x- ^( T& \, c+ p7 \
9 l& B# E7 X% Q* N/ ^# E" b  X

13. Huang, LE,Gu J,Schau M,andBunn HF. Regulation of hypoxia-inducible factor 1 is mediated by an O 2 -dependent degradation domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci USA 95:7987-7992,1998.1 H" }! c- |+ h" ^# k+ ~0 X0 T+ k
5 ^8 q% y" T4 V  {. F; B6 w: W

14. Ivan, M,Kondo K,Yang H,Kim W,Valiando J,Ohh M,Salic A,Asara JM,Lane WS,andKaelin WG, Jr. HIF- targeted for VHL-mediated destruction by proline hydroxylation: implications for O 2 sensing. Science 292:464-468,2001.- m7 I( y( h3 ?/ u* f) n
$ s  a4 {1 S/ _  y7 f

3 U& K+ M5 Q6 I- z: F+ [1 f" \/ o
15. Jaakkola, P,Mole DR,Tian YM,Wilson MI,Gielbert J,Gaskell SJ,Kriegsheim AV,Hebestreit HF,Mukherji M,Schofield CJ,Maxwell PH,Pugh CW,andRatcliffe PJ. Targeting of HIF- to the von Hippel-Lindau ubiquitylation complex by O 2 -regulated prolyl hydroxylation. Science 292:468-472,2001.
1 Q0 S+ y2 d# y3 w1 l

1 {$ ^/ J& ~3 J8 k3 }( C
16. Kallio, PJ,Wilson WJ,O'Brien S,Makino Y,andPoellinger L. Regulation of the hypoxia-inducible transcription factor 1 by the ubiquitin-proteasome pathway. J Biol Chem 274:6519-6525,1999.2 n/ f1 P8 n& u* |& v5 u# v
( W  j! U9 r! x% Q
- Y* W! z) c: E2 p
" ~" \& ?' _+ [* M9 |" d0 a
17. Kietzmann, T,Fandrey J,andAcker H. Oxygen radicals as messengers in oxygen-dependent gene expression. News Physiol Sci 15:202-208,2000.3 M( [. i8 x; Q, e
- j9 w$ X+ x$ K* F: s6 B7 i

18. Lee, PJ,Jiang BH,Chin BY,Iyer NV,Alam J,Semenza GL,andChoi AMK Hypoxia-inducible factor-1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia. J Biol Chem 272:5375-5381,1997.
2 E+ K* E+ P4 `( V+ c0 W  {

19. Luo, X,Christie NA,McLaughlin MA,Belcastro R,Sedlackova L,Cabacungan J,Freeman BA,andTanswell AK. H 2 O 2 mediates O 2 toxicity in cultured fetal rat distal lung epithelial cells. Free Radic Biol Med 26:1357-1368,1999.) D+ A: a' P: K
# f- {: ~3 L8 y! M( D2 {7 C. @
. A5 g# L4 J, M2 _
& z& f* T# O, z) {& F8 O, F
20. Min, JH,Yang H,Ivan M,Gertler F,Kaelin WG, Jr,andPavletich NP. Structure of an HIF-1-pVHL complex: hydroxyproline recognition in signaling. Science 296:1886-1889,2002.
. |/ {2 m. v, a2 W

21. Okada, H,Woodcock-Mitchell J,Mitchell J,Sakamoto T,Marutsuka K,Sobel BE,andFujii S. Induction of plasminogen activator inhibitor type 1 and type 1 collagen expression in rat cardiac microvascular endothelial cells by interleukin-1 and its dependence on oxygen-centered free radicals. Circulation 97:2175-2182,1998.% ]; J" m" ?& S; a5 b  q
7 X8 x4 @+ f+ N8 J* s" k

22. Panchenko, MV,Farber HW,andKorn JH. Induction of heme oxygenase-1 by hypoxia and free radicals in human dermal fibroblasts. Am J Physiol Cell Physiol 278:C92-C101,2000.

7 |) u, Y- _9 A, }

23. Reinheckel, T,Sitte N,Ullrich O,Kuckelkorn U,Davies KJ,andGrune T. Comparative resistance of the 20S and 26S proteasome to oxidative stress. Biochem J 335:637-642,1998.! E. F% w- K  f/ a

24. Reinheckel, T,Ullrich O,Sitte N,andGrune T. Differential impairment of 20S and 26S proteasome activities in human hematopoietic K562 cells during oxidative stress. Arch Biochem Biophys 377:65-68,2000.

+ ]' T: y  D2 z4 }9 V9 f5 B

25. Salceda, S,andCaro J. Hypoxia-inducible factor 1 (HIF-1 ) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem 272:22642-22647,1997.
9 X/ P) ?+ J; Q# l) x; `
: ^6 O7 a- V) y- M; o2 l0 ^* x, S8 H

26. Sandau, KB,Fandrey J,andBrune B. Accumulation of HIF-1 under the influence of nitric oxide. Blood 97:1009-1015,2001.1 o6 B- x, \5 A/ }

) X0 [- t4 Y! N
/ r) q$ f% r, k; c+ p
27. Semenza, GL. HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol 88:1474-1480,2000.2 W+ c8 E9 Z8 C# L& i: \( x2 g' n

" u  x' ~7 J6 D) R. l

28. Semenza, GL,andWang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol 12:5447-5454,1992., ?$ `+ P/ ]9 R

+ r$ O1 _2 @/ ~. }- C2 H4 a
29. Stolk, J,Hiltermann TJ,Dijkman JH,andVerhoeven AJ. Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. Am J Respir Cell Mol Biol 11:95-102,1994.

; C4 k3 u  ~7 O
30. Tyrrell, RM,andBasu-Modak S. Transient enhancement of heme oxygenase 1 mRNA accumulation: a marker of oxidative stress to eukaryotic cells. Methods Enzymol 234:224-235,1994." Y, I) u: z, h. V4 q: o  E2 ]

* @& u% i* F2 e* ^
31. Wang, GL,Jiang BH,Rue EA,andSemenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O 2 tension. Proc Natl Acad Sci USA 92:5510-5514,1995., w& v) C: ~. }6 r& P

: \9 L9 F- [/ p2 t
, w8 `5 y. @, H) Z, _+ W
32. Wang, GL,andSemenza GL. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J Biol Chem 268:21513-21518,1993.

33. Wang, GL,andSemenza GL. Purification and characterization of hypoxia-inducible factor 1. J Biol Chem 270:1230-1237,1995.

4 |1 Z' y, x- g
34. Wenger, RH,andGassmann M. Oxygen(es) and the hypoxia-inducible factor-1. Biol Chem 378:609-616,1997.8 r) W  Q0 t! m( o

3 w' Y! k8 D8 @
35. Wenger, RH,Rolfs A,Marti HH,Guenet JL,andGassmann M. Nucleotide sequence, chromosomal assignment and mRNA expression of mouse hypoxia-inducible factor-1. Biochem Biophys Res Commun 223:54-59,1996.

) K8 O; h$ N6 c4 z/ ^
$ w. ]. E$ X- ~9 m! j0 c
36. Wiesener, MS,Turley H,Allen WE,Willam C,Eckardt KU,Talks KL,Wood SM,Gatter KC,Harris AL,Pugh CW,Ratcliffe PJ,andMaxwell PH. Induction of endothelial PAS domain protein-1 by hypoxia: characterization and comparison with hypoxia-inducible factor-1. Blood 92:2260-2268,1998.$ B- Y5 C, B4 b. e1 v& B4 Q

4 B- I  o" |7 l9 U/ Y/ h
+ J, _% X3 a6 k4 @1 S* e/ e
37. Yang, ZZ,andZou AP. Transcriptional regulation of heme oxygenases by hypoxia-inducible factor-1 in renal medullary interstitial cells. Am J Physiol Renal Physiol 281:F900-F908,2001.7 d; o! ~6 ?3 l. z4 x$ i
; b0 e- b0 m8 P9 ?! y# x. J, b
; A5 `" j8 a0 I5 K' p
9 P4 @: Q0 h5 V' p
38. Yang, ZZ,andZou AP. Homocysteine increases TIMP-1 expression and cell proliferation associated with NADH oxidase in rat mesangial cells. Kidney Int 63:1012-1020,2003.
; P8 F6 |' b# l6 b. _- O" D
% X+ l2 m# i& D1 k" x' U- o: V

39. Yu, AY,Frid MG,Shimoda LA,Wiener CM,Stenmark K,andSemenza GL. Temporal, spatial, and oxygen-regulated expression of hypoxia-inducible factor-1 in the lung. Am J Physiol Lung Cell Mol Physiol 275

818-L826,1998.- s. X2 i; F. x! z' p

40. Zhang, DX,Zou AP,andLi PL. Ceramide-induced activation of NADPH oxidase and endothelial dysfunction in small coronary arteries. Am J Physiol Heart Circ Physiol 284:H605-H612,2003.9 F, v' B: P, |+ R
* k- L! w, X; s, K9 H) W9 c, l
# Z7 @$ Y9 d# n  {3 u

41. Zhu, H,Jackson T,andBunn HF. Detecting and responding to hypoxia. Nephrol Dial Transplant 17, Suppl 1:3-7,2002.
. ]2 Z  A, \; `0 E

8 i5 Q9 \7 D, q- b
42. Zhu, H,Qiu H,Yoon HW,Huang S,andBunn HF. Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells. Proc Natl Acad Sci USA 96:14742-14747,1999.. t* v* V' H! M" q, y1 Z
: v" V5 B6 m5 y# m9 |8 j; F5 V
; l0 Y  C+ w0 r! {
6 {2 Q5 w$ T0 A, B% r; u
43. Zou, AP,Billington H,Su N,andCowley AW, Jr. Expression and actions of heme oxygenase in the renal medulla of rats. Hypertension 35:342-347,2000.: ~  L4 x8 O  \  }- Y

& U# a9 ]$ K" P/ d" Y
44. Zou, AP,Li N,andCowley AW, Jr. Production and actions of superoxide in the renal medulla. Hypertension 37:547-553,2001.0 p4 x# `2 i  O/ l2 s! f  @; f
$ e2 M# W0 w- `0 x  d3 P- \
6 H  w/ Q% U, D: J
9 o9 J3 x  d+ u6 m3 k* t7 I
45. Zou, AP,Wu F,andCowley AW, Jr. Protective effect of angiotensin II-induced increase in nitric oxide in the renal medullary circulation. Hypertension 31:271-276,1998.
7 P3 E- G6 i  U
- ^" {: h) {* }& D2 O0 e$ T

46. Zou, AP,Yang ZZ,Li PL,andCowley AW, Jr. Oxygen-dependent expression of hypoxia-inducible factor-1 in renal medullary cells of rats. Physiol Genomics 6:159-168,2001.

收藏0 分享0 顶0 踩0