atypical mechanism of nf-κb activation during reoxygenation stress in microvascular endothelium: a...

Original Contribution

ATYPICAL MECHANISM OF NF-�B ACTIVATION DURINGREOXYGENATION STRESS IN MICROVASCULAR ENDOTHELIUM: A ROLE

FOR TYROSINE KINASES

RAMESH NATARAJAN, BERNARD J. FISHER, DREW G. JONES,1 and ALPHA A. FOWLER IIICenter for Vascular Inflammation Research, Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine,

Virginia Commonwealth University, Richmond, VA, USA

(Received 4 June 2002; Accepted 13 June 2002)

Abstract—The transcription factor nuclear factor �B (NF-�B) regulates genes that contribute to acute inflammatoryreactions in cytokine-activated endothelium. Tumor necrosis factor activates NF-�B through serine phosphorylation,induced by inhibitor �B kinases (IKK), and subsequent degradation of inhibitor �B (I�B). In contrast to cytokine stress,our studies show that oxidative stress, generated by exposure to hypoxia followed by reoxygenation (H/R), failed toactivate IKK in human microvascular endothelial cells (HMEC-1). We report an alternative mechanism for NF-�Bactivation during H/R stress without I�B� degradation. This mechanism involves activation of protein tyrosine kinases(PTK) that phosphorylate I�B� with peak phosphorylation occurring after 30 min of reoxygenation. Involvement ofPTK was reinforced by the demonstration that the PTK inhibitor, herbimycin A, prevented H/R-mediated NF-�Bactivation. Tyrosine phosphorylation alters the association between I�B� and NF-�B with sufficient intensity to allowtransient NF-�B translocation to the cell nuclei within 45 min of onset of reoxygenation stress. Immunofluorescenceimaging of NF-�B protein reveals it to be shuttled between the nucleus and cytoplasm within 90 min of reoxygenation.Furthermore, I�B� appears to be associated with NF-�B during the nucleo-cytoplasmic shuttling and is thus protectedfrom degradation. Overall, these studies suggest that tyrosine phosphorylation of I�B� represents a proteolysis-independent mechanism of NF-�B activation that can be targeted for preventing H/R-mediated injury without affectingnormal inflammatory responses. © 2002 Elsevier Science Inc.

Keywords—NF-�B, I�B�, Peroxynitrite, Endothelium, Hypoxia, Reoxygenation, Tyrosine kinase, Mechanism, Freeradicals

INTRODUCTION

Ischemia/reperfusion- or hypoxia/reoxygenation (H/R)-associated injury contributes significantly to morbidityand mortality of patients with hemorrhagic shock,early organ transplant dysfunction, stroke, and periph-eral vascular insufficiency [1–3]. Recent research sug-gests that parenchymal microvascular endothelium isactivated during reoxygenation/reperfusion [4,5]. Dur-ing reperfusion, activated endothelium is primarilyresponsible for recruitment of neutrophils to sites of

injury. Subsequent activation of neutrophils at injurysites in turn amplifies acute inflammatory responses.

Resting endothelium transcribes genes producing keyproducts (e.g., nitric oxide, prostacyclin, and adenosine)that maintain nonthrombogenic surfaces of vessel walls,thus reducing the degree of interaction with circulatingcellular elements of blood. These molecules prevent ad-hesion and maintain normal blood flow [6]. Endothelialcells activated by exposure to inflammatory mediatorssuch as tumor necrosis factor-� (TNF-�) or H/R undergophenotypic changes resulting in transcription of genesthat contribute to acute inflammation. Among the keygenes activated are adhesion molecules (e.g., E-selectin,intercellular adhesion molecule-1) and chemotactic cy-tokines (e.g., Interleukin-8) that promote neutrophil teth-ering and transendothelial migration [7]. Transcriptionalregulation of genes central to inflammation requires

1Recipient, Virginia Thoracic Society Research Fellowship TrainingAward.

Address correspondence to: Dr. Alpha A. Fowler III, Professor ofMedicine, Chairman, Division of Pulmonary and Critical Care Medi-cine, Virginia Commonwealth University, Department of Internal Med-icine, Box 980050, Richmond, VA 23298, USA; Tel: (804) 828-3558;Fax: (804) 828-3559; E-Mail: [email protected].

Free Radical Biology & Medicine, Vol. 33, No. 7, pp. 962–973, 2002Copyright © 2002 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/02/$–see front matter

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binding of transcription factor nuclear factor �B (NF-�B) to cognate sequences in the promoters of thesegenes. Unlike other transcription factors that require denovo synthesis, mature NF-�B protein is held inactive inthe cytoplasm to a family of inhibitor proteins designatedinhibitor �B (I�B). Upon activation by a variety ofstimuli (e.g., cytokines), I�B proteins are phosphorylatedby inhibitor �B kinases (IKK) at serines 32 and 36. I�Bproteins are subsequently ubiquitinated and degraded byproteasomes [8,9], a phenomenon that leads to exposureof nuclear translocation signals on NF-�B molecules anddirects translocation of NF-�B to the nucleus wherepromoter binding occurs [10,11].

Recent evidence suggests that NF-�B drives tran-scription of I�B� protein, a process that may function asa negative feedback loop [12]. Furthermore, newlyformed I�B� migrates to nuclei disrupting the DNA-binding activities of NF-�B [12]. While this pathway ofNF-�B activation is observed following cytokinestresses, recent work suggests that alternative pathwayslead to NF-�B activation with H/R stress. Canty et al.[13] and Boyle et al. [7] demonstrated NF-�B activationwithout I�B� degradation following oxidant stress inhuman umbilical vein endothelial cells. These investiga-tions suggested that this “stimulus-specific mechanism”of NF-�B activation works independently of I�B� deg-radation, possibly requiring tyrosine phosphorylation[7,13]. However, in myeloid U397 cells and HeLa cells,Mukhopadhyay et al. [14] showed that activation ofNF-�B via a tyrosine phosphatase inhibitor (pervana-date) requires both tyrosine phosphorylation of I�B� andits subsequent degradation. We have recently demon-strated that NF-�B activation occurs in reoxygenatinghuman microvascular endothelial cells (HMEC-1) with-out degradation of I�B� [15]. However, the mechanismof activation of NF-�B induced by reoxygenation stressin HMEC-1 remains undefined.

In this study we demonstrate that HMEC-1 exposed toH/R stress exhibit rapid onset of NF-�B activation. Un-like the response to cytokine stress (e.g., TNF-�), NF-�Bactivation following exposure to H/R stress occurs bytyrosine phosphorylation of I�B�, without significantdegradation of I�B�. Moreover, translocation of NF-�Bto HMEC-1 nuclei and its subsequent migration back tothe cytoplasm was associated with simultaneous I�B�migration to and from HMEC-1 nuclei. These findingssuggest that a novel signaling pathway regulates theunique shuttling of NF-�B over the course of H/R stress.

MATERIALS AND METHODS

Reagents and kits

Polyacrylamide Readygels were obtained from Biorad(Hercules, CA, USA). Electrophoretic mobility shift kits

were obtained from Promega (Madison, WI, USA). Ster-ile tissue culture plasticware was obtained from Corning(Corning, NY, USA). Culture media was obtained fromGIBCO-Invitrogen (Carlsbad, CA, USA). Specialtygases were obtained from BOC gases (Murray Hill, NJ,USA). The polyclonal antibodies to I�B� (sc-371) andNF-�B (p65 subunit; sc-109), monoclonal antibody tophosphotyrosine (sc-7020), and oligonucleotide probefor NF-�B were obtained from Santa Cruz Biotechnol-ogy (Santa Cruz, CA, USA). Hypoxia chambers (Mod-ular Incubator Chamber) were obtained from Billups-Rothenberg Inc. (Del Mar, CA, USA). Nitrocellulose andImmobilon P membranes (PVDF) were obtained fromMillipore (Bedford, MA, USA). The kit for determiningcell viability (Live/Dead), Alexa fluor 546 conjugatedgoat antirabbit IgG (H � L), and 4,6-diamidino-2-phe-lylindole HCl (DAPI) were obtained from MolecularProbes (Eugene, OR, USA). Peroxynitrite (ONOO�) wasobtained from Alexis Corp. (San Diego, CA, USA). ApH-neutralized negative control solution, containingidentical concentrations of nitrite, H2O2, and NaCl as theONOO� stock solution, and NaOH at 10 �M (AlexisCorp.) was employed as a negative control. All otherchemicals and reagents were obtained from SigmaChemical Co. (St. Louis, MO, USA).

Endothelial cell culture

The HMEC-1 cell line utilized for this study wasobtained from the Centers for Disease Control and Pre-vention (CDC), Atlanta, GA, USA. The cell line resultedfrom transfection of human dermal microvascular endo-thelial cells with a PBR-322-based plasmid containingthe coding region for the SV40 A gene product and largeT antigen. The cell line was immortalized by Dr. EdwinAdes, Mr. Fransisco J. Candal of the CDC, and Dr.Thomas Lawly of Emory University and was designatedHMEC-1. HMEC-1 were cultured under sterile condi-tions and maintained in medium MCDB-131 supple-mented with 10% fetal bovine serum, 1% penicillin/streptomycin, 2 mM glutamine, hydrocortisone (1 �g/ml), and epidermal growth factor (10 ng/ml) under a 5%CO2 atmosphere at 37°C.

Establishing hypoxia/reoxygenation conditions

Hypoxia/reoxygenation conditions were generated aspreviously described [15]. Briefly, HMEC-1 cultureswere exposed to hypoxic environments (3% O2) for 6 h.Repeated measures revealed that within 30 min of initi-ating hypoxia, media oxygen concentrations diminishedto 3% and remained unchanged for periods of up to 24 h.Our studies showed that the partial pressures of oxygen

963Atypical NF-�B activation

in hypoxic culture medium fell to a nadir of 30 � 3mmHg within 30 min. Fresh culture medium added at theoutset of reoxygenation had partial pressures of oxygenof 150 � 4 mmHg. At 6 h, reoxygenation stress wasinitiated by opening chambers and returning cells toatmospheric oxygen tensions (21% O2) without addi-tional oxygen supplementation. At the initiation ofreoxygenation, fresh culture medium was exchanged andreoxygenating cells incubated at 37°C in 5% CO2 and95% air.

Assessing HMEC-1 viability duringreoxygenation stress

Viability of HMEC-1 subjected to hypoxia/reoxygen-ation was assessed as described previously [15] using atwo-color fluorescence (LIVE/DEAD, MolecularProbes) assay. Our studies showed that HMEC-1 sub-jected to reoxygenation stress as described maintainedgreater than 99% viability.

Preparation of whole cell extracts (WCE) and nuclear/cytosolic extracts

For preparation of WCE, reoxygenating or normoxiccontrol HMEC-1 in 35 mm dishes were washed oncewith ice-cold PBS and lysis buffer [1� phosphate-buff-ered saline (PBS), 1% Nonidet P-40, 0.5% deoxycholate,0.1% SDS, 1 mM PMSF, and 40 �g/ml each of: pepsta-tin A, aprotinin, and leupeptin] added. Cell lysates werepassed through a #21 gauge needle, and centrifuged for20 min at 14,000 � g. The protein concentration of thesupernatant (WCE) was assessed and stored at �80°C.

For preparation of nuclear/cytosolic extracts, reoxy-genating or normoxic control HMEC-1 were washedonce (PBS) and exposed to buffer A (10 mM HEPES, 10mM KCl, 100 �M EDTA, 100 �M EGTA, 2 mM NaF,2 mM Na3VO4, 100 �M PMSF, 1 mM PMSF, and 40�g/ml each of: pepstatin A, aprotinin, and leupeptin) for15 min at 4°C. Cells were policed into 1.5 ml tubes, lysed(Igepal CA-630, 0.05%), and centrifuged (13,000 � g).The supernatants were used as cytosolic extracts. Nu-clear pellets were washed once with buffer A and thenresuspended in buffer B (20 mM HEPES, 390 mM NaCl,1 mM EDTA, 1 mM EGTA, 2 mM NaF, 2 mM Na3VO4,2 mM PMSF), protein concentration assessed, and sam-ples stored at �80°C.

Inhibitor �B � Western blot analysis

Whole cell extracts (10 �g protein), cytosolic extracts(10 �g protein), or nuclear extracts (15 �g) and lowmolecular weight markers were resolved by SDS poly-

acrylamide gel electrophoresis (10%) and electrophoreti-cally transferred to nitrocellulose/polyvinylidene fluoridemembranes (PVDF, 0.45 �m pore size). Immunodetec-tion was performed using a primary I�B� antibody (San-ta Cruz Biotechnology) and the Renaissance WesternBlot Chemiluminescence Reagent Plus (Perkin ElmerLife Sciences Inc., Boston, MA, USA). All membraneswere stained with Ponceau S solution (0.2% wt/vol in 1%acetic acid, Sigma Chemical Co.) to ensure equal loadingand transfer of proteins.

Electrophoretic mobility shift assay (EMSA)

EMSA was performed as previously described [15].Briefly, 5 �g of nuclear protein was incubated in abinding reaction with [32P] end-labeled double-strandedNF-�B oligonucleotide. DNA-binding reactions wereperformed at room temperature for 20 min in 50 mM Tris(pH 7.5), 50 mM NaCl, 1 mM dithiothreitol, 0.05%Igepal, and 2 �g poly d[I-C]. Samples were resolved on6% polyacrylamide gels at 100 V and imaged by auto-radiography. Specific NF-�B binding was verified by acompetitive EMSA with 100-fold excess unlabeled dou-ble-stranded NF-�B oligonucleotide in the binding reac-tion prior to the addition of the [32P] end-labeled oligo-nucleotide.

Inhibitor �B kinase � (IKK �) assay

Reoxygenating or normoxic control HMEC-1 in 35mm dishes were washed with ice-cold PBS and homog-enized in cold whole-cell extract buffer (40 mM Tris-HCl, pH 8.0, 6 mM EDTA, 6 mM EGTA, 300 mM NaCl,0.3 mM Na3VO4, 0.1% Nonidet P-40, 10 mM NaF, 10mM p-nitrophenyl phosphate, 10 mM �-glycerophos-phate, 1 mM DTT, 0.1% (v/v) 2-mercaptoethanol, 1 mMPMSF, 1 �M Microcystin-LR, and 40 �g/ml each of:pepstatin A, aprotinin, and leupeptin). Cell lysates werecentrifuged (13,700 � g, 10 min) and supernatants incu-bated with 1 �g rabbit anti-IKK� antibody at 4°C for 3 h.Antigen-antibody complexes were immunoprecipitatedovernight (4°C) by adding protein A-agarose beads. Im-munoprecipitates were recovered by centrifugation andwashed sequentially for 10 min with: (i) whole-cell ex-tract buffer with 500 mM NaCl � 3; (ii) kinase assaywash buffer (50 mM Tris-HCl and pH 7.4, 40 mMNaCl) � 1; and, (iii) kinase assay buffer (20 mMHEPES, pH 7.4, 10 mM �-glycerophosphate, 2 mMMgCl2, 2 mM MnCl2, 10 mM p-nitrophenyl phosphate,10 mM NaF, 0.3 mM Na3VO4, and 1 mM DTT) � 1.IKK� activity was determined by incubating immuno-precipitates in a reaction mixture containing kinase assaybuffer, 0.5 �g glutathione S-transferase-inhibitor �B�

964 R. NATARAJAN et al.

(GST- I�B�) protein, 10 �M ATP, and 5 �Ci [�-32P]ATP for 30 min at 30°C. Reactions were terminated byboiling in 2� SDS-PAGE sample buffer for 5 min.Phosphorylated GST-I�B� was resolved on 10% SDS-PAGE. The gels were dried and autoradiographed andthe radioactivity incorporated in GST-I�B� determinedby densitometry (Eastman Kodak Company [New Ha-ven, CT, USA] EDAS 120 system using 1D Imageanalysis software).

Identification of tyrosine-phosphorylated I�B�

Reoxygenating or normoxic control HMEC-1 werewashed once with PBS and homogenized in cold lysisbuffer (40 mM Tris-HCl, pH 8.0, 6 mM EDTA, 6 mMEGTA, 300 mM NaCl, 0.3 mM Na3VO4, 10 mM �-glyc-erophosphate, 1 mM dithiothreitol, 0.1% Igepal-CA, 10mM NaF, 10 mM p-nitrophenyl phosphate, 0.1% (v/v)2-mercaptoethanol, 1 mM PMSF, and 40 (�g/ml) each ofpepstatin A, aprotinin, and leupeptin). Cell lysates werecentrifuged (13,700 � g, 10 min), and supernatants in-cubated with 1 �g anti-I�B� antibody at 4°C for 3 h.Antigen-antibody complexes were immunoprecipitatedovernight at 4°C by adding protein A-agarose beads.Beads were recovered by centrifugation, washed, andboiled for 5 min with 2� reducing sample buffer. Pro-teins were resolved on 10% SDS-PAGE gel, electro-transferred to nitrocellulose membrane and probed withantiphosphotyrosine monoclonal antibody (0.5 �g/ml).Immunodetection was performed using the RenaissanceWestern Blot Chemiluminescence Reagent Plus as de-scribed above. Blots were stripped as described by Kauf-mann et al. [16] and reprobed with I�B� as a loadingcontrol.

Immunofluorescence for NF-�B and I�B�

To investigate intracellular trafficking of NF-�B andI�B�, HMEC-1 were grown on sterile glass coverslips.Cell cultures on coverslips were maintained under nor-moxic conditions or exposed to hypoxic conditions (3%O2) for 6 h followed by reoxygenation (21% O2) for 45and 90 min. Culture media were aspirated and cells werefixed in 3.7% paraformaldehyde in PBS for 10 min at4°C. Cells were permeablized by exposure to 0.15%Triton X-100 in PBS for 5 min at 4°C, washed withTriton/PBS, and blocked with 0.5% BSA in PBS for 30min at room temperature. Cells were incubated eitherwith rabbit anti-I�B� or anti-NF-�B (p65 subunit) poly-clonal antibodies. For detection, cells were subsequentlyincubated with Alexa Fluor 546 conjugated goat antirab-bit IgG (H � L). Finally, cells were stained with DAPI(1 �g/ml) to visualize nuclei. Fluorescence imaging of

HMEC-1 was performed using an Olympus model IX70inverted phase microscope (Olympus America, Melville,NY, USA) outfitted with an IX-FLA fluorescence obser-vation system equipped with a TRITC filter cube (530–560 nm excitation, 590–650 nm emission, ChromaTechnology Corp., Brattleboro, VT, USA) through anUplan FI objective (40�) as described previously [15].Fluorescence images were digitized and captured by aMagnaFire digital camera (Optronics, Goleta, CA,USA).

Statistical analysis

Mean values were calculated from data obtained fromthree or more separate experiments. The significance wasassessed by Student’s t-test. Statistical significance wasconfirmed at a p value � .05. A minimum of threeindependent experiments was used to confirm observa-tions.

RESULTS

Hypoxia/reoxygenation stress activates NF-�B withoutI�B� degradation in HMEC-1

Activation/degradation profile of NF-�B and I�B�in HMEC-1 subjected to cytokine stress was initiallydetermined. HMEC-1 in 35 mm dishes were exposedto TNF-� (10 ng/ml) for 15, 30, 45, 60, and 90 min.Cells were harvested and whole-cell extracts and nuclear/cytosolic extracts purified as described in Methodsabove. Figure 1 shows the results of a representativeexperiment where serial assessments of NF-�B andI�B� were performed by EMSA and Western blotanalysis, respectively. Cytokine stress with TNF-�dramatically increased nuclear NF-�B DNA binding[(15-fold, p � .001), Fig. 1A]. NF-�B protein re-mained in the nuclei of cytokine-stressed HMEC-1 forthe duration of the experiment (90 min). Western blotanalysis using whole-cell extracts showed that cyto-kine stress also produced rapid degradation (15 min)of I�B� (Fig. 1C, 85%, p � .001). I�B� levels wererestored by de novo transcription as described previously[12]. Unlike cytokine stimulus (Fig. 1A), reoxygen-ation stress initiated following 6 h of hypoxia-inducedminimal NF-�B activation after 15 min (Fig. 1B) withno observable I�B� degradation (Fig. 1C). Nuclearfactor-�B DNA-binding activity peaked 45 min follow-ing onset of reoxygenation (Fig. 1B, 5-fold, p � .001)and, thereafter, rapidly returned to control levels 90min after onset of reoxygenation. The rapid flux ofNF-�B binding was not accompanied by any significantchange in I�B� protein levels as detected by Westernblot analysis using whole-cell extracts (Fig. 1C). How-


Fig. 1. Effect of TNF-� (10 ng/ml) and H/R stress on NF-�B and I�B� in HMEC-1. HMEC-1 cells were grown to confluence in 35mm dishes. (A) Cells were exposed to media alone (c) or media with TNF-� (10 ng/ml) for 15, 30, 45, 60, and 90 min. (B) HMEC-1were maintained under normoxic conditions (n) or exposed to hypoxic conditions (3% O2) for 6 h followed by reoxygenation at roomair (21% O2) for 0, 15, 30, 45, 60, and 90 min. Cells were harvested for preparation of nuclear/cytosolic extracts and whole-cell lysates.Nuclear extracts (5 �g) were used for electrophoretic mobility shift assays [(A) and (B)] using labeled double-stranded consensus


ever, Western blots for I�B� from cytosolic extracts ofHMEC-1 subject to H/R stress demonstrated a slightdecrease in I�B� protein after 45 min of reoxygenation(Fig. 1D). This coincided with the peak in NF-�B DNA-binding activity seen in Fig. 1B. Since we had initiallydemonstrated that there was no change in I�B� proteinlevels, this decrease could be accounted for by nuclearmigration of I�B�. Indeed, Western blots from nuclearextracts of HMEC-1 subject to H/R stress showed ele-vated nuclear I�B� protein levels (Fig. 1E). Therefore,these results suggest that I�B� moves in a conjointfashion with NF-�B following onset of reoxygenationstress.

Hypoxia/reoxygenation stress does not activateinhibitor �B kinase � (IKK�)

NF-�B activation occurs following endothelial cellexposure to a variety of stimuli including cytokines,

viruses, and oxidants [11]. Most NF-�B-activating stim-uli induce degradation of I�B� via phosphorylation oftwo serine residues in the N-terminal portion of I�B�[11]. The kinases that elicit serine phosphorylation aretermed inhibitor �B kinases (IKK) [7,9]. While severalIKK isoforms exist, IKK� is the predominant kinaseactivated during cytokine stresses. In these studies wedetermined the time course for IKK� activation inHMEC-1 cells following exposure to H/R and cytokinestresses. As seen in Fig. 2A, IKK� activity was un-changed over a period of 90 min following onset ofreoxygenation. Exposure to cytokine stress dramaticallyincreased IKK� activity over the same time period (Fig.2B). Increased IKK� activity was evident as early as 7.5min (3.6-fold, p � .001), with a peak in activationoccurring after 30 min (7-fold, p � .001). Cytokinestimulus with TNF-� coincident with reoxygenationstress did not significantly alter the activation pattern of

Fig. 2. Hypoxia/reoxygenation stress does not activate inhibitor �B kinase � (IKK�). HMEC-1 were grown to confluence in 35 mmdishes. (A) Cells were maintained under normoxic conditions (*) or exposed to hypoxic conditions (3% O2) for 6 h followed byreoxygenation at room air (21% O2) for 15, 30, 45, and 90 min. (B) Cells were treated with media alone (#) followed by TNF-� (10ng/ml) for 7.5, 15, 30, 45, and 90 min, or (C) exposed to hypoxic conditions (3% O2) for 6 h plus TNF-� (10 ng/ml) for 7.5, 15, 30,45, and 90 min following onset of reoxygenation. Cells were harvested and IKK� kinase assay performed as described in Methods.

NF-�B as described in Methods. Competition assay using cold unlabeled oligonucleotide in TNF-�-treated sample (30 min) is shown in (A) (cold).The specifically shifted band is indicated as NF-�B. Nonspecific bands are designated n.s. The free probe is also indicated with an arrow. (C)Whole-cell lysates (10 �g), (D) cytosolic extracts (10 �g), or (E) nuclear extracts (15 �g) were separated by SDS-polyacrylamide gel electrophoresis(10%) and electrophoretically transferred to polyvinylidene fluoride membranes (Immobilon P, 0.45 �m pore size). Immunodetection was performedusing a primary I�B� antibody and the Renaissance Western Blot Chemiluminescence Reagent Plus. All experiments were performed in triplicate.A media/normoxic control (c/n) is shown in lane 1 of the representative assay.


IKK� activation (Fig. 2C). However, the combined ef-fect of H/R � TNF-� caused a decrease in IKK� activityat 45 and 90 min more markedly than with TNF-� alone.Therefore while H/R stress in these studies seems tohasten the downregulation of TNF-�-activated IKK�,our results suggest that initial IKK� activation per se isunaffected by H/R stress. Thus, NF-�B activation inHMEC-1 following onset of reoxygenation stress occursin an IKK-independent manner.

Protein tyrosine kinase inhibitors suppress NF-�Bactivation during reoxygenation stress in HMEC-1

Several reports have recently stressed alternativepathways for NF-�B activation [7,13,14]. These hypoth-eses suggest a “stimulus-specific mechanism” for NF-�Bactivation, independent of I�B� degradation, involvingI�B� tyrosine phosphorylation. To examine this hypoth-esis, HMEC-1 were subjected to H/R stress in the pres-ence or absence of protein tyrosine kinase (PTK) inhib-itors, herbimycin A, staurosporine, and genistein. Cellswere harvested after varying periods of H/R stress andnuclear extracts purified as described above. Preincuba-tion with herbimycin A (2 �M) abolished NF-�B acti-vation (95% decrease, p � .001) during reoxygenationstress (Fig. 3A). The PTK inhibitor genistein (100 �M)also significantly suppressed (50% decrease, p � .001)NF-�B activation (Fig. 3B) 45 min after onset of reoxy-

genation. Staurosporine in the nanomolar range is apowerful PTK inhibitor, while higher concentrations willalso inhibit protein kinase C. As shown in Fig. 3B,staurosporine abolished NF-�B activation (95% de-crease, p � .001) during reoxygenation stress. Thesepharmacological data suggest that activation of one ormore PTKs is critical for NF-�B activation that occursduring reoxygenation stress in HMEC-1.

Peroxynitrite activates NF-�B in HMEC-1 cells

In the setting of H/R, simultaneous generation of reac-tive nitrogen and oxygen species (nitric oxide and superox-ide anion) by vascular endothelium produces a biradicalreaction resulting in the formation of peroxynitrite anion(OONO�). To determine whether peroxynitrite activatesNF-�B, HMEC-1 were exposed to authentic peroxynitrite.As seen in Fig. 4, peroxynitrite produced a concentration-dependent increase in NF-�B activation (5-fold, p � .001with 10 �M and 12-fold, p � .001 with 150 �M). NF-�Bactivation in these studies occurred without I�B� degrada-tion, suggesting that peroxynitrite-induced NF-�B activa-tion was similar to that found in reoxygenation stress.

I�B� is tyrosine phosphorylated during reoxygenationstress in HMEC-1

HMEC-1 were treated with authentic peroxynitrite(10 �M) for 1 h in the presence or absence of the PTK

Fig. 3. Protein tyrosine kinase (PTK) inhibitors suppress NF-�B activation during reoxygenation stress in HMEC-1. HMEC-1 weregrown to confluence in 35 mm dishes. Cells were exposed to hypoxic conditions (3% O2) for 6 h followed by reoxygenation at roomair (21% O2) for 15, 45, and 90 min in the absence or presence of (A) herbimycin A (2 �M) and (B) staurosporine (50 nM) or genistein(100 �M). Cells were harvested for preparation of nuclear extracts. Nuclear extracts (5 �g) were used for electrophoretic mobility shiftassays using labeled double-stranded consensus NF-�B as described in Methods. All experiments were performed in triplicate. Anegative control (�) is shown in lane 1 of the representative assay.


inhibitor staurosporine (50 nM), or exposed to H/Rstress in the presence or absence of the PTK inhibitorstaurosporine (50 nM). I�B� was immunoprecipitatedas described above at various time periods, followedby Western blot analysis with antiphosphotyrosinemonoclonal antibody. As seen in Fig. 5A, authenticperoxynitrite induced tyrosine phosphorylation ofI�B� (3-fold, p � .005). The observed induction wasblocked by preincubation with staurosporine (50 nM)

for 30 min. The status of tyrosine phosphorylation ofI�B� was also changed after H/R stress. Fig. 5Bshows that tyrosine phosphorylation of I�B� is detect-able 15 min following onset of reoxygenation, withpeak phosphorylation occurring after 30 min (�2-fold, p � .005). Tyrosine phosphorylation of I�B�was subsequently restored to control levels 90 minafter onset of reoxygenation. Peak tyrosine phosphor-ylation of I�B� during reoxygenation stress (30 min)was blocked with staurosporine (50 nM), implying akey involvement of PTKs. The time frame of phos-phorylation events suggests a potential role for this“stimulus-specific mechanism” to occur prior to acti-vation of NF-�B and its subsequent nuclear transloca-tion. These findings also suggest that NF-�B activa-tion during reoxygenation stress appears to arise dueto tyrosine phosphorylation of I�B� by as yet uniden-tified PTKs.

NF-�B and I�B� protein exhibit a unique pattern ofcellular trafficking following onset ofhypoxia/reoxygenation stress in HMEC-1

As shown in Fig. 1B, H/R stress induced NF-�Bactivation and translocation 45 min after onset ofreoxygenation stress. However, under conditions ofreoxygenation, NF-�B becomes rapidly inactivated, a

Fig. 4. Peroxynitrite activates NF-�B in HMEC-1. HMEC-1 weregrown to confluence in 35 mm dishes. Cells were treated with mediaalone or with 10 �M and 150 �M authentic peroxynitrite for 1 h. Cellswere harvested for preparation of whole-cell/nuclear extracts. Nuclearextracts (5 �g) were used for electrophoretic mobility shift assays usinglabeled double-stranded consensus NF-�B as described in Methods.Whole cell extracts (10 �g) were used for Western blot analysis ofI�B� as described in Fig. 1. All experiments were performed intriplicate. A normoxic control (*) is shown in lane 1 of the represen-tative assay.

Fig. 5. I�B� is tyrosine phosphorylated during reoxygenation stress in HMEC-1. HMEC-1 were grown to confluence in 35 mm dishes.(A) Cells were treated with media alone or media containing authentic peroxynitrite (10 �M) for 1 h in the presence or absence ofstaurosporine (50 nM). (B) Cells were maintained under normoxic conditions (*) or exposed to hypoxic conditions (3% O2) for 6 hin the presence or absence of staurosporine (50 nM) followed by reoxygenation in room air (21% O2) for 0, 15, 30, 45, and 90 min.Cells were lysed and I�B� immunoprecipitated as described in Methods. Immunoprecipitated proteins were resolved on 10%SDS-PAGE gel, electrotransferred to nitrocellulose membrane, and probed with antiphosphotyrosine monoclonal antibody (0.5 �g/ml).Blots were stripped and reprobed for I�B� as described. All experiments were performed in triplicate. A normoxic control (*) is shownin lane 1 of the representative assay.


pattern distinct from that observed with cytokinestress. To examine this phenomenon we used immu-nofluorescence microscopy to histologically localizethese two protein moieties. DAPI was used as a coun-terstain to identify nuclei in these studies (data notshown). As shown in Figs. 6A and 6D, I�B� andNF-�B, respectively, are predominantly extranuclearin resting, normoxic HMEC-1. Following 6 h of hyp-oxia (3% O2) and 45 min of reoxygenation stress (21%O2), NF-�B resides in a nuclear location (Fig. 6E), aswould be expected from EMSA studies presentedabove in Fig. 1B. Interestingly, I�B� is simulta-neously present in nuclei 45 min after onset of reoxy-genation stress (Fig. 6B). Furthermore, 90 min after

onset of reoxygenation, both I�B� and NF-�B arecolocalized and have relocated to HMEC-1 cytoplasm(Figs. 6C and 6F). These results along with Westernblot analysis from Fig. 1 suggest that I�B� moves ina conjoined fashion with NF-�B as it shuttles to andfrom HMEC-1 nuclei following the onset of reoxy-genation stress.

DISCUSSION

Reoxygenation or reperfusion of previously ischemictissue results in a series of cellular events indistinguish-able from an acute inflammatory response [17]. Theabrupt return of circulation to vascular beds commonly

Fig. 6. NF-�B and I�B� exhibit a unique pattern of cellular trafficking following onset of hypoxia/reoxygenation stress in HMEC-1.HMEC-1 were grown on sterile glass coverslips. Cells were maintained under normoxic conditions [(A) and (D)] or exposed to hypoxia(3% O2) for 6 h followed by reoxygenation in room air (21% O2) for 45 [(B) and (E)] and 90 min [(C) and (F)], respectively. Cellswere fixed in 3.7% paraformaldehyde in PBS for 10 min at 4°C. Cells were permeablized by exposure to 0.15% Triton X-100 in PBSfor 5 min at 4°C, washed with Triton/PBS and blocked with 0.5% BSA in PBS for 30 min at room temperature. Cells were incubatedeither with rabbit anti-I�B� [(A)–(C)] or anti-NF-�B (p65 subunit) polyclonal antibodies [(D)–(F)]. For detection (indicated byarrows), cells were subsequently incubated with Alexa fluor 546 conjugated goat antirabbit IgG (H � L) and visualized using anOlympus model IX70 inverted phase microscope outfitted with an IX-FLA fluorescence observation system through Uplan FI objective(40�). Fluorescence images were digitized and captured by a MagnaFire digital camera.


results in significant vascular injury and the frequentdevelopment of organ failure [1–3]. Several reports sug-gest that reperfusion injury arises from generation ofROS following reintroduction of oxygen after ischemicevents in a variety of different cell types [18,19]. Wehave previously demonstrated extremes of oxygenationin our model system, thereby genuinely providing anenvironment of substantially changed partial pressures ofoxygen [15]. Reoxygenation stress as modeled thereinduced significant increases in microvascular endothe-lial oxidative activity [15]. Our current study focuses onthe mechanisms underlying H/R stress in microvascularendothelium in our previously established model system.

Transcription factor NF-�B is activated by diversestimuli, and this event plays an essential role in endo-thelial cell activation. The pathways leading to NF-�Bactivation are uniquely stimulus and cell-type specific.We report here that human microvascular endotheliumsubjected to H/R stress produces NF-�B activation with-out I�B� degradation (Fig. 1). IKK�, a kinase widelyaccepted to be activated by cytokine stress, was neitherstimulated nor inhibited by H/R stress (Fig. 2A), andtherefore was not involved in this alternative mechanismof NF-�B activation. While the phenomenon of NF-�Bactivation without I�B� degradation has recently beenreported [13,14,20], the mechanism of activation remainsunknown. Several workers have proposed that tyrosinephosphorylation of I�B� by PTKs is the alternativemechanism for NF-�B activation [14,21,22]. Singh et al.[23] used the tyrosine phosphatase inhibitor pervanadateand demonstrated in ML-1a, U937, and HeLa cells thatsite-specific tyrosine phosphorylation of I�B� negativelyregulates its inducible phosphorylation and degradation,thus preventing NF-�B activation. In contrast, Imbert etal. [22] showed that pervanadate produced NF-�B acti-vation through tyrosine phosphorylation without degra-dation of I�B�. In the current study, H/R stress producedtyrosine phosphorylation of I�B�, with peak phosphor-ylation occurring 30 min after the onset of reoxygenation(Fig. 5). Increased I�B� tyrosine phosphorylation wassubsequently followed by translocation of NF-�B to nu-clei from cytoplasm, a molecular event that occurred 45min after onset of reoxygenation (Fig. 1B). Our studiesfurther showed that PTK inhibitors, herbimycin A, stau-rosporine, and genistein, blocked NF-�B activation andI�B� tyrosine phosphorylation. These results suggestthat PTK-mediated I�B� tyrosine phosphorylation is es-sential for NF-�B activation in H/R stress of HMEC-1(Figs. 3 and 5).

In the setting of H/R stress in vascular endothelium,Beckman et al. showed that peroxynitrite anion(OONO�) forms spontaneously by simultaneous gener-ation of NO and O2

�• [24]. We have recently demon-strated that reactive oxygen species (ROS) are produced

immediately upon reoxygenation of HMEC-1 cells ex-posed to hypoxia (3% O2) for 6 h [15]. In unpublishedstudies, we have observed increased inducible nitric ox-ide synthase mRNA and activity, and increased produc-tion of NO during the 6 h exposure to hypoxic conditions(data not shown). Taken together, these results suggestthat NO and ROS (and therefore peroxynitrite) are si-multaneously generated at the onset of reoxygenation inHMEC-1. While the detrimental effect of OONO� iswidely reported [24,25], its effects on modulation of cellsignaling pathways are relatively unknown. Zouki et al.showed that micromolar concentrations of OONO� ac-tivated ERK in neutrophils by the Ras/Raf-1/MEK signaltransduction pathway [26]. More recently, Matata et al.showed that OONO� regulates cytokine production inhuman mononuclear cells promoting NF-�B activationin a dose-dependent manner [25]. In this study, we dem-onstrate that authentic peroxynitrite activates NF-�Bwithout I�B� degradation (Fig. 4). Moreover, H/R stressin HMEC-1 induced tyrosine phosphorylation of I�B�with peak phosphorylation events occurring 30 min afteronset of reoxygenation. The peak in I�B� tyrosine phos-phorylation preceded translocation of NF-�B toHMEC-1 nuclei, which occurred 45 min following onsetof reoxygenation. These results suggest that reoxygen-ation stress, perhaps through the generation of peroxyni-trite, induces PTK activity, which subsequently leads toNF-�B activation without I�B� degradation.

Several reports suggest that tyrosine phosphorylationof I�B� protects the inhibitory molecule from degrada-tion [13,14,20]. While Singh et al. [23] showed thattyrosine phosphorylation of I�B� prevented NF-�B ac-tivation, others demonstrate that this phosphorylationevent leads to NF-�B activation [13,14]. Potent activa-tors of NF-�B, such as TNF-�, Interleukin-1, or lipo-polysaccharide, induce rapid degradation of I�B� byphosphorylation of serines 32 and 36 on I�B�, polyu-biquitination of I�B� primarily at lysines 21 and 22, andfinally degradation by the 26S proteasome [27]. Inhibi-tors of the 26S proteasome efficiently block nucleartranslocation of NF-�B, indicating that neither phosphor-ylation nor ubiquitinylation is sufficient to dissociateI�B� from NF-�B [28–32].

Tyrosine 42 of I�B� is located in close proximity toserines 32 and 36 in the N-terminal regulatory region[30]. Therefore, it is unlikely that phosphorylation oftyrosine 42 alone is sufficient for release of the inhibitorI�B� from NF-�B. Tyrosine phosphorylated peptides arespecifically recognized by SH2 domains, which arestructural motifs found in several cytoplasmic signalingproteins [33]. To this extent, Imbert et al. [22] showedthat phosphorylated tyrosine 42 residues on I�B� inter-act with SH2 domain-containing proteins. Beraud et al.showed in T cells that I�B�, newly phosphorylated at


tyrosine 42 following pervanadate exposure, associateswith the regulatory subunit of PI3-kinase, a het-erodimeric lipid and protein kinase composed of a SH2domain-containing regulatory subunit [34]. While thistheory was not examined in the current study, our West-ern blot (Fig. 1E) and immunofluorescence (Fig. 6) datashow that I�B� moves in a conjoined fashion withNF-�B in posthypoxic HMEC-1 nuclei. Therefore, wepropose that, following H/R stress in HMEC-1, tyrosinephosphorylation of I�B� likely alters its association withNF-�B.

While not resulting in physical dissociation from NF-�B, tyrosine phosphorylation of I�B� exposes nuclearlocalization signals on NF-�B molecules, a prerequisiteevent for its nuclear translocation. Our studies suggestthat NF-�B then translocates to HMEC-1 nuclei bound toI�B�. In a time-sensitive fashion in posthypoxic nuclei,protein tyrosine phosphatases may dephosphorylate ty-rosine 42 on I�B�, permitting the “conventional” reas-sociation of I�B� with NF-�B, which then masks nu-clear translocation signals. Resumption of a“prephosphorylation” configuration likely results in shut-tling of NF-�B/I�B� complexes back to HMEC-1 cyto-plasm by a previously described negative feedback loop[12]. The reappearance of I�B� in HMEC-1 cytoplasmcontemporaneous to NF-�B 90 min after onset of reoxy-genation stress (Fig. 6) supports this hypothesis.

NF-�B is typically coupled to various I�B proteins inresting cells. Only I�B� in the I�B family of proteins hasa tyrosine residue at position 42 [34]. Other I�B proteinslack a site homologous to tyrosine 42 of I�B�. There-fore, by being bound to I�B�, NF-�B may specificallyrespond to stimuli that induce tyrosine phosphorylation,thereby permitting cell type- and stimulus-specific regu-lation.

In summary, our results show that reoxygenationstress in human microvascular endothelial cells activatesNF-�B independent of I�B� degradation. Furthermore,we have provided new evidence for an alternative path-way that may be targeted for prevention of H/R-mediatedinjury without impacting normal inflammatory re-sponses.

Acknowledgements — This research was supported by funds from theNational Institutes of Health (HL-61359 and HL-10355) and the Vir-ginia Thoracic Society.

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ABBREVIATIONS

BSA—bovine serum albuminDAPI—4,6-diamidino-2-phelylindole HClHMEC-1—human microvascular endothelial cell-1H/R—hypoxia/reoxygenationI�B�—inhibitor �B�IKK�—inhibitor �B kinase �NF-�B—nuclear factor �BPBS—phosphate-buffered salinePTK—protein tyrosine kinasePTP—protein tyrosine phosphatasesROS—reactive oxygen speciesTNF-�—tumor necrosis factor-�


atypical mechanism of nf-κb activation during reoxygenation stress in microvascular endothelium: a...

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