Am J Physiol Cell Physiol 308: C673–C683, 2015. First published February 4, 2015; doi:10.1152/ajpcell.00367.2014.

Inhibitor-␬B kinase attenuates Hsp90-dependent endothelial nitric oxide synthase function in vascular endothelial cells Mohan Natarajan,1 Ryszard Konopinski,2 Manickam Krishnan,1 Linda Roman,3 Alakesh Bera,4 Zheng Hongying,1 Samy L. Habib,5 and Sumathy Mohan1 1

Department of Pathology, University of Texas Health Science Center at San Antonio, San Antonio, Texas; 2Department of Molecular Biology, Cancer Center Institute, Warsaw, Poland; 3Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas; 4Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas; and 5South Texas Veterans Health System, San Antonio, Texas Submitted 17 November 2014; accepted in final form 2 February 2015

Natarajan M, Konopinski R, Krishnan M, Roman L, Bera A, Hongying Z, Habib SL, Mohan S. Inhibitor-␬B kinase attenuates Hsp90-dependent endothelial nitric oxide synthase function in vascular endothelial cells. Am J Physiol Cell Physiol 308: C673–C683, 2015. First published February 4, 2015; doi:10.1152/ajpcell.00367.2014.—Endothelial nitric oxide (NO) synthase (eNOS) is the predominant isoform that generates NO in the blood vessels. Many different regulators, including heat shock protein 90 (Hsp90), govern eNOS function. Hsp90-dependent phosphorylation of eNOS is a critical event that determines eNOS activity. In our earlier study we demonstrated an inhibitor-␬B kinase-␤ (IKK␤)-Hsp90 interaction in a high-glucose environment. In the present study we further define the putative binding domain of IKK␤ on Hsp90. Interestingly, IKK␤ binds to the middle domain of Hsp90, which has been shown to interact with eNOS to stimulate its activity. This new finding suggests a tighter regulation of eNOS activity than was previously assumed. Furthermore, addition of purified recombinant IKK␤ to the eNOS-Hsp90 complex reduces the eNOS-Hsp90 interaction and eNOS activity, indicating a competition for Hsp90 between eNOS and IKK␤. The pathophysiological relevance of the IKK␤-Hsp90 interaction has also been demonstrated using in vitro vascular endothelial growth factormediated signaling and an Ins2Akita in vivo model. Our study further defines the preferential involvement of ␣- vs. ␤-isoforms of Hsp90 in the IKK␤-eNOS-Hsp90 interaction, even though both Hsp90␣ and Hsp90␤ stimulate NO production. These studies not only reinforce the significance of maintaining a homeostatic balance of eNOS and IKK␤ within the cell system that regulates NO production, but they also confirm that the IKK␤-Hsp90 interaction is favored in a high-glucose environment, leading to impairment of the eNOS-Hsp90 interaction, which contributes to endothelial dysfunction and vascular complications in diabetes. inhibitor-␬B kinase-␤; endothelial nitric oxide synthase function; nitric oxide; endothelial cells; high glucose

is characterized by the reduced bioavailability of nitric oxide (NO) in the vasculature (16, 18). Impaired endothelial NO synthase (eNOS) activity and regulation play a major role in the vascular complications of diabetes. Optimal generation of NO by eNOS is dependent on several factors, including availability of the substrate L-arginine and the cofactor tetrahydrobiopterin (11). Activation of eNOS is a complex process and may be regulated by transcriptional and posttranslational mechanisms, site-specific phos-

ENDOTHELIAL DYSFUNCTION

Address for reprint requests and other correspondence: S. Mohan, Dept. of Pathology, Univ. of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229 (e-mail: [email protected]). http://www.ajpcell.org

phorylation of eNOS, protein-protein interactions, prosthetic groups, and Ca2⫹ and calmodulin (1). eNOS has been shown to interact with several regulatory proteins, such as heat shock protein (Hsp90), caveolin-1, G protein-coupled receptors, NO-interacting protein, dynamin-2, and porin (1, 9). In endothelial cells (ECs), tetrahydrobiopterin and Hsp90 have been shown to be important effectors in regulating eNOS activity. Hsp90 is known to regulate Ca2⫹dependent dissociation of eNOS from caveolin-1, enzyme activation, maturation, and trafficking, followed by the Aktdependent phosphorylation of eNOS at Ser1177 (human eNOS) or Ser1179 (bovine eNOS). Phosphorylation of eNOS at Ser1177 or Ser1179 is a key posttranslational modification associated with stimulation of NO synthesis (12). The binding of Hsp90 to eNOS ensures the transition from the early Ca2⫹-dependent to the late phosphorylation-dependent activation of eNOS. Failure of this binding has been demonstrated to cause eNOS uncoupling and an increase in eNOS-dependent superoxide anion production. In our earlier study, in a chronic high-glucose (HG) environment, we found that production of inhibitor-␬B kinase-␤ (IKK␤) in ECs was enhanced and that the activity of IKK␤ is dependent on the IKK␤-Hsp90 interaction (22). In parallel studies of NO production in ECs in a HG environment, Kim et al. (14) suggested that HG-mediated endothelial dysfunction involves activation of IKK␤, which inhibits insulin receptor substrate (IRS-1)/phosphatidylinositol 3-kinase signaling and, thereby, attenuates NO production. The requirement of Hsp90 for activation of the IKK complex, consisting of IKK␣, -␤, and -␥, has been demonstrated in cancer and transformed cell lines (4, 6, 8, 24, 25). The existence of an IKK␤-Hsp90 interaction in primary vascular ECs was demonstrated for the first time in our earlier report (22). Its influence on HG-mediated NO generation in ECs has also been investigated. The Hsp90 proteins, named for their 90-kDa average molecular mass, belong to a group of highly conserved molecular chaperones that account for 1–2% of all proteins in cells under nonstress conditions. Hsp90 proteins can be found in the cytosol, nucleoplasm, endoplasmic reticulum, and mitochondria (5). In vertebrates, two major cytosolic isoforms of Hsp90, Hsp90␣ and Hsp90␤, (which are ⬃85% identical in amino acid sequence), are common (21). Hsp90␣ is inducible under stress conditions, while Hsp90␤ is constitutively expressed (15). While both of these isoforms are involved in cellular processes, including signal transduction and intracellular transport, and have clients that include protein kinases, transcription factors such as p53, and steroid hormone receptors, they also

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have distinct responses to different conditions/inducers (15, 17, 21, 27). The relationship between the increased expression of IKK␤, its preferential binding to Hsp90, and its influence on eNOS activity in a HG environment is unknown. The data presented in this study identify the precise binding domain of IKK␤ on Hsp90 and the pathophysiological relevance of this interaction in the regulation of NO signaling. The data advance our knowledge of how IKK␤ interacts with the two cytosolic isoforms of Hsp90. The new insights obtained from this study will help in development of modified therapeutic modalities involving IKK␤ blocking that could potentially lessen the severity of vascular complications of diabetes.

were obtained after centrifugation of the tissue homogenates at 100,000 g at 4°C (Optima MAX-TL ultracentrifuge, Beckman Coulter). Then the IKK␤flox/flox mice were bred, and 8- to 10-wk-old mice were euthanized to purify ECs from the aorta (Cell Biologics). Cells released from aortae by digestion were incubated with antiplatelet endothelial cell adhesion molecule (PECAM-1) antibody following addition of magnetic beads precoated with secondary antibody. Cells released from the magnetic beads were washed and cultured on a gelatin-coated culture dish. Initial-passage cells were gently scraped with a sterile blunt cell scraper before they were plated. ECs were confirmed to be 93.38% pure by PECAM-1 staining, vascular endothelial cadherin expression, and acetylated LDL (DilAc-LDL) uptake, and purified ECs were used for transfection experiments. Generation of FLAG-Tagged Deletion Constructs of Hsp90 and Full-Length IKK␤-Enhanced Yellow Fluorescent Protein Fusion Plasmid

MATERIALS AND METHODS

Cell Culture Differentiated adult human aortic ECs (HAECs; Invitrogen, Carlsbad, CA), bovine aortic ECs (BAECs; Clonetics, San Diego, CA), and mouse aortic ECs (MAECs; Cell Biologics, Chicago, IL) were cultured as described in our previous report (23). Briefly, cells were grown in MCDB-131 medium (Sigma, St. Louis, MO) containing fetal bovine serum (FBS; Hyclone, Kansas City, KS) and enriched with 250 ng/ml fibroblast growth factor (PeproTech, Rocky Hill, NJ), 20 ng/ml epidermal growth factor (PeproTech), 1 ␮g/ml hydrocortisone (Sigma), and 100 U/ml penicillin and 100 mg/ml streptomycin (Media-Tech, Herndon, VA). In general, 10% FBS is used for revival. For routine maintenance, only 5% FBS was used. Prior to treatment conditions, the cells were incubated in medium with reduced (2%) FBS concentration for ⱖ16 h. Cells from passages 4 –7 were used for all experiments. Treatment Conditions BAECs, HAECs, or MAECs were maintained in MCDB-131 medium supplemented with 10% FBS until 18 h before treatments, when this medium was replaced with medium with reduced (5%) FBS concentration. Basal growth medium containing 5.5 mM glucose was considered the normal glucose control. For HG conditions, cells were incubated with additional 25 mM ␣-D-(⫹)-glucose for ⬎48 h or up to 2 wk. Prolonged incubation in 25 mM glucose was chosen to reflect hyperglycemia (which could be achieved at the postprandial level and spikes higher after a meal and persists longer based on diet, differential responses, and treatment plans), which contributes to the severity of endothelial dysfunction in vivo. Also, endothelial responses could be compared with earlier studies, since this concentration has been widely used (14, 16, 18). In addition, cells were incubated with 25 mM mannitol and used as an osmotic control. For vascular endothelial growth factor (VEGF) induction, cells cultured with 2% FBS-containing medium for 18 h were incubated with a fresh aliquot of VEGF [a recombinant human dimerized VEGF-A165 (PeproTech)] at 25–50 ng/ml for 15 min. Animal Experiments All procedures were performed in accordance with National Institutes of Health guidelines, with protocol approval by the University of Texas Health Science Center at San Antonio Animal Care and Use Committee. Type 1 diabetic Ins2Akita mice (stock no. 003548, Jackson Laboratory) and IKK␤flox/flox mice (procured from Dr. Michael Karin, University of California, San Diego) were bred in-house and genotyped using tail biopsies. Heterozygous diabetic (Ins2Akita) mice (4 – 6 mo old) with blood glucose levels ⬎400 mg/dl were euthanized, and thoracic and abdominal aortae were dissected. After the blood was removed, the vessels were homogenized using tissue protein extraction buffer (1⫻ HEPES-based buffer, pH 7.5). Clear supernatants

To subclone the mammalian IKK␤ coding sequence, the insert containing the complete open reading frame was excised using EcoRI and ApaI restriction enzymes from the pCR3-FLAG vector (obtained from Dr. Hiroyasu Nakano, Tokyo, Japan). It was subsequently ligated into the pEYFP-C1 plasmid (Clontech) expressing yellow fluorescent protein (YFP) (22). To keep the proper reading frame, the plasmid was reconstructed by changing the position of an EcoRI site using a synthetic linker/adapter. The pCR3-FL-Hsp90 fusion plasmid was created by subcloning the Hsp90 reading frame from the pGEX vector (obtained from Dr. Richard Venema, Medical College of Georgia, Augusta, GA) into the pCR3-FL2 vector bearing two repeats of the FLAG tag sequence upstream of the cloning site. The deletion mutants generated consisted of full-length FLAG-tagged Hsp90, COOH-terminal deletion (⌬BX, aa 643–711), middle domain with COOH-terminal deletion (⌬EX, aa 304 –711), and NH2-terminal deletion (⌬dN, aa 1–328; and ⌬EE, aa 304 –328). All constructs were validated for their orientation and intact reading frame by standard molecular methods. Overexpression of siRNA Specific for Hsp90 For transient transfection, cells at passage 4 and in the exponentially growing phase were trypsinized and made up as a single-cell suspension. Cells at a density of 2 ⫻ 106 cells in 100 ␮l of R buffer (Life Technology, Carlsbad, CA) were electroporated with human Hsp90AA1 (Hsp90␣) siRNA (10 ␮M) and/or Hsp90AB1 (Hsp90␤) siRNA (10 ␮M) constructs. Scrambled siRNA construct was also transfected and used as the negative control. Transfection efficiency was determined by cotransfection of siGLO Red (RNA-induced silencing complex-free fluorescently labeled nontargeting oligonucleotide) reagent. Electroporation was carried out by the Neon transfection system (Life Technologies) at a pulse rate of 2, a pulse voltage of 1,400 V, and a pulse width of 20 ms. An equal number of transfected cells were plated on gelatin-coated 100-mm culture dishes for protein expression and a six-well plate for quantitative PCR (qPCR) analysis and incubated at 37°C for 36 – 48 h prior to lysis. Deletion of IKK␤ From ECs of IKK␤flox/flox Mice by Overexpression of pBS598-EF1␣EGFP/Cre The presence of the Lox-P site in ECs purified from aortae of IKK␤flox/flox mice were first confirmed by PCR using genomic DNA as the template and primers that were used for genotype screening of tail DNA biopsies. For transient transfection, cells at passage 4 and in the exponentially growing phase were trypsinized and made up as a single-cell suspension. Cells at a density of 2 ⫻ 106 cells in 100 ␮l of R buffer (Life Technologies) were electroporated with pBS598EF1␣EGFP/Cre (Addgene, Cambridge, MA) as described above. After 48 h of transfection, cells were harvested, and the lysates were

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immunoblotted using anti-IKK␤ antibody. Cells were plated in gelatin-coated 100-mm culture dishes and incubated for 36 – 48 h, and NO was measured by 4-amino-5-methylamino-2=,7=-difluorofluorescein diacetate [DAF-FM (DA)] using fluorescein-activated cell sorting (FACS) (see below). To confirm the effect of IKK␤ deletion, its downstream target of NF-␬B DNA binding activity was measured in these transfected cells induced with 25 mM glucose using methods as we reported earlier (19).

eNOS activity measurements. eNOS activity was determined using the hemoglobin capture assay. NO formation was measured at 23°C in pH 7.4 buffer containing 50 mM Tris·HCl, 100 mM NaCl, and 200 ␮M CaCl2, as described elsewhere (13). Rates of NO synthesis were determined using an extinction coefficient of 60 mM⫺1·cm⫺1 at 401 nm.

Hsp90 Messenger RNA Expression by qPCR Analysis

Cells cultured in complete growth medium containing 5% FBS were transiently transfected with siRNA specific for Hsp90␣ or Hsp90␤, or mouse IKK␤flox/flox cells were transfected with pBS598EF1␣EGFP/Cre, as described above. After 48 h of transfection, cells were treated with a recombinant human dimerized VEGF-A165 (25 ng/ml) for 15 min. To determine the selective influence of HG on NO production, cells were left with endogenous levels of other essential cofactors required for NO generation and compared with untreated controls. Cells were washed three times with Krebs Ringer-bicarbonate buffer (Sigma-Aldrich) and further incubated with 0.5 ␮M DAF-FM (DA) for 30 min at 37°C. Cells were then trypsinized, collected, and resuspended in Krebs Ringer-bicarbonate buffer containing 2% FBS. Cells were analyzed on a FACS Contour-II (BD Biosciences) at 100,000 events, and data are represented as changes in mean fluorescence intensity (488-nm excitation wavelength and 530-nm emission wavelength) at the single-cell level. In each sample, the mean fluorescence intensity of the analyzed cells was determined after the cell population was gated by forward- and side-scatter light signals as recorded on a dot plot. Data were analyzed using FACSDiva software (BD Biosciences). To exclude any intracellular background fluorescence signal, cells incubated with DAF-FM (DA) alone, in parallel with glucose-treated cells, were used as the basal level of fluorescence. Fluorescence data calculated by subtraction of unstained cells from stained cells are expressed as percentage of mean fluorescence.

TaqMan gene expression assay (Life Technologies) was used to quantify mRNA expression levels. Briefly, total RNA extracted by the PureLink RNA isolation kit (Life Technologies) was reverse-transcribed using the SuperScript III RT-PCR kit (Life Technologies) in a reaction mixture containing random hexamer primers (for mRNA RT-PCR). Then qPCR was performed in triplicate with reactions containing amplified cDNA and TaqMan primers in Universal Master Mix without AmpErase uracil N-glycosylase following the manufacturer’s protocol (Applied Biosystems, Carlsbad, CA). All mRNA data are presented relative to 18S expression obtained from the same samples. Fold expression was calculated from the triplicate of comparative threshold (CT) values following the 2⫺⌬⌬CT method. Immunoprecipitation and Western Blotting Whole cell lysates were precleared by incubation with protein A and protein G-coupled Sepharose 4B beads for ⬃1 h at 4°C, as described elsewhere (21). The cleared lysates were transferred into a fresh tube and incubated with 1–3 ␮g of immunoprecipitating primary antibody, rabbit (Rb) anti-human Hsp90, Rb anti-human eNOS, Rb anti-human IKK␤, or preimmune IgG (Millipore, Billerica, MA) followed by protein A ⫹ protein G-coupled Sepharose 4B beads. The immune complexes were separated by electrophoresis on a 10% SDS-polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) for immunoblot analysis. Blots were blocked for 45 min using a 3% low-fat milk solution (PBS containing 3% low-fat milk and 0.1% Tween 20) and further incubated overnight at 4°C in blocking solution containing a primary antibody: Rb anti-human eNOS (1:1,000 dilution), Rb anti-human IKK␤ (1:1,000 dilution), or Rb anti-human Hsp90 (1:1,000 dilution). Bound antibodies were detected with peroxidase-conjugated secondary antibodies (1:10,000 dilution) and visualized using chemiluminescence Western blot detection reagent (HyGlo quick spray, Denville Scientific). ␤-Actin expression was used as internal loading control. For immunoprecipitation experiments, the efficacy of each immunoprecipitation was normalized by probing the membrane with the antibody to determine whether an equal amount of respective antigen was immunoprecipitated. NO Measurements Protein expression and purification. Bovine eNOS was expressed and purified as previously described (19), with a few modifications. After sonication, the cell lysate was applied to a 50-ml DEAESepharose column (Sigma-Aldrich, St. Louis, MO) equilibrated in 20 mM Tris·HCl, pH 7.4, 100 mM NaCl, 0.1 mM EDTA, 0.1 mM DTT, and 10% glycerol (buffer B). Protein was eluted with 0 –500 mM NaCl in buffer B. eNOS eluted at ⬃250 –300 mM NaCl. eNOS-containing fractions were loaded onto a 30-ml 2=5=-ADP-Sepharose affinity column equilibrated in buffer B, washed with 300 ml of buffer B, and eluted with 50 ml of buffer B containing 500 mM NaCl and 5 mM 2=-AMP. Calmodulin was prepared as described elsewhere (30). Spectrophotometric methods. The molar protein concentrations for eNOS were determined on the basis of heme content via reduced CO difference spectra, where the extinction coefficient was 100 mM⫺1·cm⫺1 for ⌬A445-470 (26). All spectral analyses were performed using a Shimadzu model 2401PC UV/visible dual-beam spectrophotometer.

NO Measurement by DAF-FM (DA) Using FACS

Steady-State Fluorescence Polarization Measurements Steady-state fluorescence polarization was measured with the multimode microplate reader PHERAstar FS spectrofluorometer (BMG LABTECH, Cary, NC) equipped with an accessory for steady-state polarization measurement. The Alexa Fluor 488 protein labeling kit was used to label recombinant Hsp90 (rHsp90) according to the manufacturer’s protocol (Invitrogen). The labeled Hsp90 (Hsp90Alexa 488) in solution was excited at 488 nm, and polarization emission was collected at 520 nm. The dimeric binding interaction between Hsp90-Alexa 488 and eNOS or Hsp90-Alexa 488 and IKK␤ and also trimeric complex formation of Hsp90-eNOS-IKK␤ were determined. In both cases, Hsp90 was kept constant at a fixed concentration of 100 nM. We measured the fluorescence polarization of the extrinsic Alexa Fluor 488 labeled with Hsp90 as the reporter for the binding study with eNOS or IKK␤ with reference to the free protein in solution. The final value represents changes in the size of the protein complexes. We also determined whether they are bound to each other and form protein-protein stable complexes. The fluorescence polarization (P) values from the two measured channels (parallel and perpendicular) were calculated by following the equation: P ⫽ 1,000*(parallel ⫺ perpendicular)/(parallel ⫹ perpendicular), where parallel and perpendicular are the intensities of the emitted light when the emission polarizers are aligned parallel and perpendicular, respectively, to the polarization of the excited light (2). RESULTS

Overexpression of Hsp90␣ and Hsp90␤ siRNA Suppresses NO Production HAECs overexpressing Hsp90␣- or Hsp90␤-specific siRNA oligomers and treated with VEGF (25 ng/ml) exhibited a

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significant reduction of VEGF-mediated NO production (Fig. 1A). Cells induced with VEGF in the absence of Hsp90specific siRNA expression showed an 80% increase in NO production compared with untreated controls (P ⬍ 0.001). The scrambled siRNA-transfected cells treated with VEGF also demonstrated a 73% enhancement of NO production (P ⬍ 0.01). When siRNA specific for Hsp90␣ was overexpressed in the cells, NO production was reduced by 54.75 ⫾ 1.12% (P ⬍ 0.001) compared with scrambled siRNA-transfected cells induced with VEGF. Similarly, in cells overexpressed with siRNA specific for Hsp90␤, the reduction of NO production was 42.42 ⫾ 1.30% (P ⬍ 0.001) compared with VEGFinduced scrambled siRNA-transfected cells. The difference of VEGF-induced NO generation between cells transfected with Hsp90␣- and Hsp90␤-specific siRNA was not significant (P ⫽ 0.269). These results clearly show an equal contribution or involvement of Hsp90␣ and Hsp90␤ in NO generation through VEGF signaling. As shown in Fig. 1B, cells incubated with 1 mM nitro-L-arginine methyl ester, an eNOS-specific inhibitor, reduced 61.26% of NO induced by VEGF, indicating the specificity of the assay. Analysis of the transfected cells by qPCR (Fig. 1C) and Western blotting (Fig. 1D) confirmed significant blocking of the mRNA and protein expression of Hsp90␣ and Hsp90␤ by overexpression of Hsp90␣- and Hsp90␤-specific siRNA. Additionally, in cells overexpressing siRNA specific for Hsp90␣, a marked increase in Hsp90␤ mRNA and a moderate level of protein overexpression, and vice versa, were observed as a compensatory response (data not shown). Binding Region of IKK␤ on the Middle Domain of Hsp90 Overlaps With That of eNOS To identify the putative binding site of IKK␤ in Hsp90, we generated a series of deletion mutants of Hsp90 to determine

which domain of Hsp90 is required for the binding of IKK␤ in intact cells. FLAG-tagged deletion mutants of Hsp90␤ include the NH2-terminal domain (⌬N, aa 1–328 deletion; and ⌬EE, aa 304 –328 deletion), middle domain (⌬EX, aa 304 –711 deletion), and COOH-terminal domain (⌬BX, aa 643–711 deletion) spanning the complete Hsp90 sequence (Fig. 2A). Cells were transfected with cDNA constructs containing enhanced YFPtagged full-length IKK␤ (EYFP-IKK␤) and one of the mutant Hsp90 constructs in pairs, and the interaction was assessed in immunoprecipitation experiments. Figure 2B, iii shows equal expression of EYFP-IKK␤, and Fig. 2B, i shows that the transfected FLAG-Hsp90 mutants were equally expressed and immunoprecipitated. As shown in Fig. 2B, ii, cotransfection of different FLAG-tagged Hsp90 deletion mutants with fulllength EYFP-IKK␤ resulted in IKK␤ coprecipitating with the NH2-terminal domain (⌬N, aa 1–328 deletion), COOH-terminal domain (⌬BX, aa 643–711 deletion), and small NH2terminal domain (⌬EE, aa 304 –328 deletion) (data not shown), but not with the middle domain (⌬EX, aa 304 –711 deletion), indicating that the binding domain does not reside in the NH2or COOH-terminal regions of Hsp90 but, rather, in the middle domain between aa 328 and 643. Interaction of IKK␤ and eNOS with Hsp90 Established Using Purified Proteins Competitive interaction of IKK␤ and eNOS with Hsp90 in a cell-free system. Addition of IKK␤ inhibits Hsp90-stimulated eNOS activity. To establish that eNOS enzyme activity, which is increased upon binding with Hsp90, is compromised in the presence of recombinant IKK␤ (rIKK␤), we determined the rate of eNOS-dependent NO synthesis before and after addition of rHsp90 and rIKK␤ to the reaction mix. The eNOS enzyme activity increased significantly (⬃40%) with addition of Hsp90 at a ratio of 1:1 compared with the basal level, indicating that

Fig. 1. Overexpression of siRNA specific for ␣- and ␤-isoforms of heat shock protein 90 (Hsp90) suppresses nitric oxide (NO) production. A: fluorescein-activated cell sorting analysis using 4-amino-5-methylamino-2=,7=difluorofluorescein diacetate [DAF-FM (DA)] fluorochrome shows induced intracellular NO levels in human aortic endothelial cells (HAECs) incubated with VEGF (25 ng/ml) compared with untreated controls (P ⬍ 0.001). Cells transiently transfected with scrambled siRNA and treated with VEGF also showed increased NO production similar to cells treated with VEGF alone. HAECs transiently transfected with Hsp90␣- or Hsp90␤-specific siRNA oligomers and treated with VEGF (25 ng/ml) exhibited significantly reduced VEGF-mediated NO production. B: VEGF-induced NO is significantly reduced by addition of 1 mM nitro-L-arginine methyl ester (L-NAME) 30 min prior to VEGF treatment. C and D: quantitative PCR (C) and Western blot (D) analysis of siRNA-transfected cells confirmed significant blocking of mRNA and protein expression of Hsp90␣ and Hsp90␤. Values are means ⫾ SD from 3 independent experiments. *P ⬍ 0.05 (by Student’s unpaired t-test).

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Fig. 2. Mapping of the Hsp90-inhibitor-␬B kinase-␤ (IKK␤) interacting domains. A: construction of Hsp90 deletion mutants, which were used to ascertain the binding site for IKK␤ on the Hsp90 domain. Deletion mutants ⌬EE (aa 304 –328 deletion), ⌬EX (aa 304 –711 deletion), ⌬BX (aa 643–711 deletion), and ⌬N (aa 1–328 deletion) were inserted into pCR2.1-FLAG plasmid and used for transient transfection. B: immunoprecipitation (IP) followed by Western blot analysis showing intact expression of deletion mutants of Hsp90 and the binding site of IKK␤ on Hsp90. Endothelial cells were cotransfected with the cDNA encoding FLAG-tagged Hsp90 mutant constructs and full-length (FL) enhanced yellow fluorescent protein (EYFP)-tagged IKK␤. i: FLAG-Hsp90 expression identifies molecular sizes of the Hsp90 mutants that have been immunoprecipitated and immunoblotted using the anti-FLAG antibody. ii: EYFP-IKK␤ complexed with mutants of FLAG-tagged Hsp90. iii: expression level of EYFP-IKK␤ in cotransfection experiments.

association with Hsp90 has the expected stimulatory effect on eNOS (Fig. 3A). However, in the presence of both Hsp90 and IKK␤, eNOS activity is decreased by 70% compared with eNOS and Hsp90 alone. eNOS activity was lower than the basal level. Polarization studies. The binding interaction of protein partners in solution measured by steady-state fluorescence polarization with Alexa Fluor 488-labeled rHsp90 and recombinant eNOS (reNOS) or Alexa 488-labeled rHsp90 and rIKK␤ clearly showed complex formation. A fixed concentration of Hsp90 (100 nM) was used in all experiments. Since the IKK␤ used in these experiments was tagged with glutathione S-transferase (GST), a control experiment was performed with GSTand Alexa 488-labeled Hsp90. This control experiment clearly demonstrated the lack of binding of GST with Hsp90. The relative polarization of a fixed concentration of rHsp90 and increasing reNOS concentrations (0 –3.5 ␮M) revealed a significant increase in complex formation (Fig. 3B). The base polarization of Alexa Fluor 488-labeled rHsp90 was taken as P0, and the difference in polarization values in the presence of eNOS or IKK␤ at increasing concentrations was calculated. The relative polarization tends to remain at the same level even after addition of 3.5 ␮M reNOS (data not shown). The maximum relative polarization for rHsp90 and rIKK␤ was only ⬃1.15 and remained at the same level even at 3.5 ␮M eNOS. These results clearly demonstrate the ability of IKK␤ to bind to Hsp90. Fusion of GST to IKK␤ did not influence the binding characteristics of IKK␤ to Hsp90, since purified GST did not show independent binding to Hsp90 (Fig. 3B). As shown in

Fig. 3C, binding of increasing concentrations of GST-IKK␤ (0 –1.2 ␮M) displaces eNOS from rHsp90. The degree of competition between IKK␤ and eNOS in binding with Hsp90 (100 nM) was measured by addition of increasing concentrations of IKK␤ or eNOS to the eNOS-Hsp90 or IKK␤-Hsp90 complex, respectively (Fig. 3C). The initial binary complexes (eNOS-Hsp90 and IKK␤-Hsp90) exhibit higher polarization values than Hsp90 alone. Addition of eNOS to the IKK␤Hsp90 complex increased polarization values, whereas addition of IKK␤ to the eNOS-Hsp90 complex decreased polarization values. Increased concentration of exogenously added purified GSTIKK␤ displaces rHsp90-bound reNOS. An immunoprecipitation approach with recombinant purified proteins biochemically validated the positive interaction of IKK␤ with Hsp90 and further confirmed that competitive binding of IKK␤ with Hsp90 displaces the Hsp90-bound eNOS. After 2 h of incubation of 10 nmol of eNOS and Hsp90 at 4°C, increasing concentrations (0 –7 nmol) of GST-tagged IKK␤ were added, and the complex was incubated for 2 h. The complex was immunoprecipitated with anti-eNOS antibody, and the immune complexes were analyzed for coprecipitating Hsp90. The presence of Hsp90 was reduced in this complex when IKK␤ concentration was increased (Fig. 3D). The lysates collected after precipitation were reprecipitated with anti-GST antibody and examined for the presence of Hsp90. As shown in Fig. 3E, addition of increasing concentrations of GST-IKK␤ reduces eNOS-bound Hsp90. The supernatants of this immune complex collected after the first immunoprecipitation were reincu-

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Fig. 3. Use of recombinant Hsp90, IKK␤, and endothelial NO synthase (eNOS) proteins in establishing the competitive interactions of eNOS and IKK␤ with Hsp90. A: hemoglobin capture assay showing kinetics of NO formation by eNOS. eNOS at 100 nM in solution was incubated in the presence of Hsp90 (100 nM) or Hsp90 ⫹ IKK-␤ (100 nM). Addition of recombinant IKK␤ significantly blocked eNOS activity. B: fluorescence polarization analysis of Hsp90, IKK␤, and eNOS interactions. Binding with eNOS (300 nM) or IKK␤ (300 nM) increased fluorescence polarization of the Alexa Fluor 488 fluorophore-labeled Hsp90. P0, polarization value of unbound Alexa Fluor-labeled Hsp90; P, polarization value of Alexa Fluor 488-labeled Hsp90 with IKK␤ or eNOS. C: degree of competition between IKK␤ and eNOS in binding with Hsp90 (100 nM) was measured by addition of increasing concentrations (0 –1.2 ␮M) of IKK␤ or eNOS to the eNOS-Hsp90 or IKK␤-Hsp90 complex, respectively. Polarization values of the IKK␤ protein added to the eNOS-Hsp90 complex were measured after 2 h of incubation at 4°C. D: immunoprecipitation assay showing that increasing concentrations of GST-IKK␤ mitigate the eNOS-Hsp90 interaction. Purified recombinant eNOS and recombinant Hsp90 were incubated with increasing concentrations (0 –7 nmol) of GST-IKK␤ for 2 h at 4°C. Interacting proteins were immunoprecipitated with anti-eNOS antibody, and the immune complexes were analyzed by Western blotting (WB). Incubation of the membrane with anti-Hsp90 antibody shows that increasing concentrations of GST-IKK␤ inhibit the eNOS-Hsp90 interaction. E: supernatants saved after immunoprecipitation with the anti-eNOS antibody were incubated with anti-GST antibody, and the immune complex was analyzed for the presence of Hsp90 using anti-Hsp90 antibody. At higher concentrations of GST-IKK␤ (7 nmol), a decrease in the eNOS-Hsp90 interaction was observed (last lane in D), concomitant with the appearance of a prominent IKK␤-Hsp90 interaction (last lane in E).

bated with anti-GST antibody to pull down the IKK␤, and bound Hsp90 reappeared in the immune complex upon addition of 7 nmol of GST-IKK␤ (Fig. 3E). This reappearance indicates that addition of GST-IKK␤ displaced Hsp90 bound to eNOS. Preferential Hsp90␣ vs. Hsp90␤ Interaction With eNOS and IKK␤ Under the Influence of HG Hsp90␣ and Hsp90␤ localized in the cytoplasm have been shown to contribute to NO generation. Independent blocking of Hsp90␣ and Hsp90␤, as shown in Fig. 1, significantly downregulates NO generation, demonstrating their contribution to eNOS activity. However, their precise role in the eNOS-IKK␤ interaction under the influence of HG is not understood. Immunoblotting of EC lysates collected after ⬎48 h in a HG

environment did not show any difference in the percent expression levels of Hsp90␣ and Hsp90␤ (Fig. 4). Cells expressed higher Hsp90␤ under both control (77.2 ⫾ 4.6%) and HG (81.03 ⫾ 2.63%) conditions relative to Hsp90␣ expression. Expression of Hsp90␣ under control (41.26 ⫾ 5.03%) and HG (41.3 ⫾ 3.15%) conditions remained at the same level without any statistical significance. However, as demonstrated in Fig. 5A, immune complexes precipitated with eNOS antibody showed reduced interaction with Hsp90␣ when the cells were under the influence of HG compared with cells in the control condition or cells exposed to mannitol. However, immune complexes precipitated with IKK␤ antibody showed enhanced interaction with Hsp90␣ (Fig. 5C). On the other hand, no difference in the eNOS-Hsp90␤ interaction was observed in a HG envi-

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IKK␤ Decreases the VEGF-Induced eNOS-Hsp90 Interaction Because of the prominent and noted sensitive responses of Hsp90␣ interacting with eNOS/IKK␤ under the influence of HG, we used the Hsp90␣-specific antibody for the following coimmunoprecipitation experiments. To demonstrate the pathophysiological significance of the IKK␤-Hsp90 interaction, physiological responses of VEGF signaling that have been demonstrated to result in an enhanced eNOS-Hsp90 interaction associated with increased NO production were studied by coimmunoprecipitation. VEGF has been shown to stimulate recruitment of Hsp90 and promote eNOS phosphorylation at Ser1177, leading to NO production (10, 20, 29). Therefore, we used VEGF as an inducer of the eNOS-Hsp90 interaction. As shown in Fig. 7A, when lysates from VEGFinduced ECs were incubated with 14 nmol of IKK␤, the Hsp90 precipitating with eNOS was significantly reduced. However, this reduction was reversed when the lysate was incubated with heat-denatured IKK␤. Physiological Significance of IKK␤ Involvement in DiabetesInduced Endothelial Dysfunction Fig. 4. Expression of Hsp90 isoforms is not altered by a high glucose (HG) environment. A: aortic endothelial cells were cultured in control medium containing 5.5 mM glucose (C) or HG (25 mM) for ⬎48 h. Cell lysates were analyzed by Western blotting, and membranes were probed with Hsp90 isoform-specific antibodies. ␣-Tubulin expression was used as loading control. B: quantitation from ⱖ3 independent experiments is represented as percent expression of Hsp90.

ronment (Fig. 6). Similarly, the IKK␤-Hsp90␤ interaction under the influence of HG also showed no significant changes with Hsp90␤ (Fig. 6), indicating the sensitivity of Hsp90␣ to a HG environment.

We previously demonstrated (7) the enhanced expression of IKK␤ in aortic ECs cultured in a HG environment. To correlate the pathophysiological significance in vivo, IKK␤ expression in aortae from Ins2Akita mice was determined. As shown in Fig. 7B, the diabetic mice exhibited significantly enhanced (2.47 ⫾ 0.18 fold, P ⬍ 0.05) IKK␤ expression compared with the wild-type control mice. The aortae of these mice exhibited significantly reduced expression of eNOS (3.03 ⫾ 0.07 fold decrease; Fig. 7C), thus demonstrating the imbalance in the expression levels of eNOS and IKK␤ in a HG environment. To determine the effect of IKK␤ deletion in ECs in blood vessels,

Fig. 5. Differential interaction of Hsp90␣ with eNOS and IKK␤ in response to HG. A: lysates of endothelial cells incubated with 25 mM glucose for ⬎48 h, immunoprecipitated with eNOS, and immunoblotted with Hsp90␣ show the eNOS-Hsp90␣ interaction. B: quantitative measurement of the eNOS-Hsp90␣ interaction from 3 independent experiments shown as fold increase compared with control. Mannitol at 25 mM or glucose at the concentration present in the basal medium (5 mM) was used as control. C: lysates of endothelial cells incubated with 25 mM glucose for ⬎48 h, immunoprecipitated with IKK␤, and immunoblotted with Hsp90␣ show the IKK␤-Hsp90␣ interaction. Mannitol (25 mM) and 5 mM glucose in the basal medium were used as controls. D: quantitative measurement of the IKK␤Hsp90␣ interaction from 3 independent experiments shown as fold increase compared with control. Blots were reprobed with respective immunoprecipitation antibodies to demonstrate that immunoprecipitation was performed with an equal amount of antigen.

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a 3.36 ⫾ 0.11 fold increment in NO production (Fig. 7G), despite their exposure to 25 mM glucose for ⬎48 h. As demonstrated in Fig. 7E, Cre-mediated transient transfection resulted in specific deletion of IKK␤ expression (without altering expression of VEGF-signaling molecules) and totally abolished HG- and TNF␣-induced downstream NF-␬B DNA binding activity, thus confirming deletion of IKK␤ (Fig. 7F). These results clearly demonstrate the role of IKK␤ in regulating NO production, thus confirming its translational potential in improving the bioavailability of NO in diabetic vessels. DISCUSSION

Fig. 6. Nondifferential interaction of Hsp90␤ with eNOS and IKK␤ in response to HG. A: lysates of endothelial cells incubated with 25 mM glucose for ⬎48 h exhibited no change in the eNOS-Hsp90␤ or IKK␤-Hsp90␤ interaction. Lysates of endothelial cells heat-shocked at 42°C for 15 and 30 min were used as positive controls to detect Hsp90 position on the membrane. C, control; M, mannitol. Reprobing of the membrane shows an equal amount of antigen being immunoprecipitated (bottom). B and C: quantitative measurement (from 3 independent experiments) of the interaction, demonstrating no significant changes in protein interactions and treatment conditions. Mannitol (25 mM) or glucose at the concentration present in the basal medium (5 mM) was used as control.

we cross-bred IKK␤flox/flox mice with Ins2Akita-Tie-2-Cre⫹ mice. Since these breeding attempts were unsuccessful (due to prenatal mortality), we purified aortic ECs from IKK␤flox/flox mice and used these cells for NO measurements. When MAECs with intact IKK␤ expression (cells from IKK␤flox/flox mice) were incubated with HG for ⬎48 h, they generated significantly reduced NO production (0.67 ⫾ 0.05 fold compared with 1.0 fold of control, P ⬍ 0.005). However, Cre-mediated deletion of IKK␤ by transfection of pBS598EF1␣EGFP/Cre in these cells reversed this effect, as shown by

The salient findings of this study are as follows: 1) by a novel mechanism, IKK␤ mitigates binding and subsequent phosphorylation of eNOS by Hsp90; 2) the putative binding site for IKK␤, determined by mapping studies with deletion mutant constructs of Hsp90, is within the binding site of eNOS in the middle domain of Hsp90; and 3) competitive binding of IKK␤ with Hsp90 in a HG environment abrogates eNOS binding to Hsp90 and, hence, plays a major role in preventing eNOS phosphorylation (Ser1177) and NO production. The fundamental understanding of structure-function relationships of eNOS with its regulators that govern the generation of NO is important in identifying targets for the design of novel therapeutic agents to improve NO bioavailability. Restoration of NO bioavailability is indispensable for alleviating endothelial dysfunction, which is a hallmark of cardiovascular complications of diabetes. Earlier studies have identified Hsp90 as a central component involved in activation/phosphorylation of eNOS (20). Overexpression of Hsp90 in BAECs has been shown to enhance basal and VEGF-stimulated eNOS phosphorylation on Ser1179, with subsequent NO release (10). More interestingly, these earlier studies clearly demonstrated that the middle domain of Hsp90 serves as a primary binding domain for eNOS. The binding domain is very similar in both isoforms of Hsp90: between aa 442 and 600 in Hsp90␣ and between aa 449 and 607 in Hsp90␤. Mapping studies performed by overexpression of EYFPtagged IKK␤ and FLAG-tagged deletion mutants of Hsp90 spanning the NH2-terminal domain (aa 1–328 and 304 –328 deletion), middle domain (aa 304 –711 deletion), and COOHterminal domain (aa 643–711 deletion) demonstrated that the middle domain, between aa 328 and 643, is the primary binding domain of IKK␤ on Hsp90. Thus the IKK␤- and eNOS-binding domains on Hsp90 could overlap. In addition, since the Akt binding site on Hsp90 has been shown to be between aa 327 and 340 (10), it is logical to assume that IKK␤ binding (between aa 328 and 643) might also interfere with Akt binding to Hsp90. Hence, an active IKK␤ binding could mitigate the productive interaction between Akt kinase and eNOS and, thus, play an important role in impairment of eNOS phosphorylation and subsequent NO production. Since the binding sites for eNOS are very similar on Hsp90␣ and Hsp90␤, it is likely that both of these isoforms could bind to IKK␤. Independent suppression of Hsp90␣ vs. Hsp90␤, as shown in Fig. 1, demonstrated reduced production of NO, indicating involvement of both isoforms in eNOS function. In cells overexpressing siRNA specific for Hsp90␣, a marked increase in Hsp90␤ mRNA and a moderate level of protein overexpression, and vice versa, were seen as a compensatory

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Fig. 7. Influence of HG on the VEGF-induced eNOS-Hsp90␣ interaction. A: endothelial cells incubated with VEGF (25 ng/ml) for 15 min showed enhanced eNOS-Hsp90␣ interaction compared with control. Recombinant IKK␤ (rIKK␤, 14 nmol) was added to the precleared cell lysate, which was then incubated for 1 h at 4°C. Heat-inactivated rIKK␤ was incubated with VEGF-induced cell lysate to demonstrate the reversal effect. B and C: expression of IKK␤ and eNOS in aortae of diabetic mice. Aortic homogenates from C57BL/6 wild-type and Ins2Akita type 1 diabetic mice were immunoblotted (50 –100 ␮g of total protein), and membranes were probed for IKK␤ (B) and eNOS (C) expression. Equal protein loading was confirmed by probing the membrane for expression of the housekeeping genes GAPDH and ␤-actin. N, number of mice. D and E: presence of the Lox-P site in endothelial cells purified from aortae of IKK␤flox/flox mice were first confirmed by PCR using genomic DNA as template (D) and immunoblot showing the specific deletion of IKK␤ after transient transfection with 2 ␮g and 3 ␮g of pBS598-EF1␣EGFP/Cre plasmid (E). IKK␣ expression was undisturbed. F: IKK␤-manipulated endothelial cells exhibit reduced downstream effector function of NF-␬B DNA binding activity. Endothelial cells purified from IKK␤flox/flox mice and transfected with pBS598-EF1␣EGFP/Cre plasmid were induced with 25 mM glucose for ⬎48 h or 10 ng/ml TNF␣ for 15 min. Nuclear proteins (5 ␮g) extracted from these cells were used to analyze activation of NF-␬B by electrophoretic mobility shift assay. G: IKK␤-manipulated endothelial cells from mouse aorta show improved NO production. Endothelial cells purified from IKK␤flox/flox mice were transfected with pBS598-EF1␣EGFP/Cre plasmid. Transfected cells were exposed to 25 mM glucose for ⬎48 h. NO measurements with DAF-FM (DA) and fluorescein-activated cell sorting analysis in these cells demonstrated enhanced NO production (3.36 ⫾ 0.11 fold increment) in the presence of HG.

response. However, this compensatory effect was not reflected in the NO production profile, probably because of the defined level of involvement of the two isoforms of Hsp90, which has a certain threshold beyond which NO production cannot be altered. Furthermore, the definite availability and the contribution of other components that are required for NO production may be limited, even though Hsp90␣ or Hsp90␤ is elevated. Involvement of Hsp90 isoforms in eNOS function could be pertinent to vascular ECs, since siRNA-specific suppression of Hsp90␤ upregulates NO2/NO3 levels in human embryonic kidney (HEK-293) cells (7). While the relevance of the use of these cells in vascular physiology and eNOS regulation is not clearly explained, this study demonstrated the significance of

phosphorylation of eNOS at different sites mediated by Hsp90␣ vs. Hsp90␤, thus up- or downregulating NO production. In our studies using purified proteins and polarization/coprecipitation techniques, it is evident that IKK␤ could displace eNOS bound to Hsp90, indicating a possible competition between eNOS and IKK␤ to bind to Hsp90. eNOS activity measurements also demonstrated this effect. Inhibition of eNOS activity could be due to 1) formation of a ternary complex, 2) formation of an IKK␤-Hsp90 complex, or 3) formation of an eNOS-IKK␤ complex (eliminating Hsp90). Our cellular coimmunoprecipitation and polarization anisotropy data strongly suggest that IKK␤ and eNOS compete for

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the binding of Hsp90. However, IKK␤ complexes with Hsp90 by replacing eNOS at a higher concentration (Fig. 3, B and C). In our earlier report (22), we demonstrated the increased temporal expression/activation of IKK␤ in HG-induced ECs. In the present study, we have demonstrated the enhanced expression of IKK␤ in vivo in the aortae of type 1 diabetic Ins2Akita mice. Reports from other laboratories have shown increased expression of IKK␤ in the blood vessels of type 2 diabetic mice (28). Also, as demonstrated in Figs. 2 and 8A, the binding domain of eNOS on Hsp90 completely overlaps with the IKK␤ binding domain. Therefore, it is possible that such a competition and displacement of eNOS from the Hsp90 binding domain could be due to increased expression of IKK␤ in a cellular or arterial system that is exposed to inducers of IKK␤, such as HG. As shown in Fig. 7, lysates from VEGF-induced ECs, when incubated with purified native IKK␤, showed a diminished eNOS-Hsp90 interaction. However, addition of heat-inactivated enzyme restored the VEGF-induced eNOSHsp90 interaction. The NO measurements performed (shown in Fig. 3A) using purified eNOS-Hsp90 complex also corroborated the negative influence of the presence of IKK␤ in the surrounding milieu. Therefore, in general, the outcome of NO production is dependent on the balance of eNOS and IKK␤ within the cytosol, which is decided by the presence of positive/negative inducers of eNOS/IKK␤. Additionally, since heat-killed enzyme reversed the effect of the eNOS-Hsp90 interaction, the kinase activity of IKK␤ could play a major role. In support of our observation, PKA-specific phosphorylation of Hsp90 has been shown to translocate Hsp90 to the exterior of the cell under the influence of HG (16). However, PKAspecific phosphorylation of eNOS has been reported to improve NO production (3). If HG could upregulate PKA activity, eNOS and Hsp90 would be phosphorylated, which will not result in diminished NO production. It is well established that HG causes reduced NO production, resulting in endothelial dysfunction in a diabetic artery. Studies establishing the regu-

lation of PKA activity in the presence of simultaneous induction of IKK␤ are therefore warranted to understand the functional interdependence of these enzymes and their impact on NO regulation. Our attempts to determine the contribution of specific isoforms of Hsp90 in generating NO in response to VEGF (Fig. 1A) revealed an active participation of Hsp90␣ and Hsp90␤. The expression of neither isoform changes under the influence of HG (Fig. 4). However, HG-treated ECs exhibited a reduced eNOS-Hsp90␣ interaction and an increased IKK␤-Hsp90␣ interaction. This differential eNOS-IKK␤ interaction was not observed with Hsp90␤, indicating that Hsp90␣ is uniquely sensitive to HG. Similar responses have been reported with different inducers (15, 17, 21, 27). The two cytosolic Hsp90 isoforms have been shown to differ in the relative efficiency with which they activate certain Hsp90 clients in yeast cells (21). However, systematic comparison of the Hsp90 isoforms with respect to cochaperone and multiple-substrate interactions indicated a nonselective distribution of endogenous Hsp90 isoforms in the cellular chaperone machinery in NIH 3T3 cells in a nonstressed condition (27), while Hsp90␣ displayed enhanced substrate preference under the influence of heat shock. It is compelling to speculate that the yet unknown mechanism that specifically stimulates Hsp90␣ under the influence of heat shock probably functions under the influence of HG as well, which results in sensitivity to the IKK␤/eNOS-Hsp90␣ interaction. The insensitivity of Hsp90␤ in complexing with eNOS/ IKK␤ under the influence of HG is unclear and needs further investigation. Reduced NO production under the influence of long-term HG incubation could be rescued by limiting IKK␤ expression, as shown in Fig. 7D, under the influence of physiological inducers, as shown in Fig. 8B. These data demonstrate the pathophysiological significance of this novel interaction and indicate a possible competition between eNOS and IKK␤ for Hsp90, which could be a reflection of the known imbalance

Fig. 8. A: overlapping binding sites of eNOS and IKK␤ on Hsp90. Amino acid position is localized on Hsp90. B: VEGF-mediated eNOS-Hsp90 interaction is impaired by HG-induced IKK␤, which competes with eNOS and downregulates the eNOS-Hsp90 interaction. AJP-Cell Physiol • doi:10.1152/ajpcell.00367.2014 • www.ajpcell.org

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between intracellular NO and reactive oxygen species within the cell under the influence of HG. ACKNOWLEDGMENTS We thank Preethi Janardhanan and Colleen Gaudette for technical help. GRANTS This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant 5 R01 DK-096119 (S. Mohan) and National Space Biomedical Research Institute Grant CA02802 through National Aeronautics and Space Administration Grant NCC 9-58-298 (M. Natarajan). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS M.N., R.K., M.K., L.R., A.B., and H.Z. performed the experiments; M.N. and S.M. analyzed the data; M.N. and S.M. interpreted the results of the experiments; M.N. prepared the figures; M.N., L.R., S.L.H., and S.M. edited and revised the manuscript; S.M. developed the concept and designed the research; S.M. approved the final version of the manuscript. REFERENCES 1. Balligand JL. Heat shock protein 90 in endothelial nitric oxide synthase signaling: following the lead(er)? Circ Res 90: 838 –841, 2002. 2. Bera A, Roche AC, Nandi PK. Bending and unwinding of nucleic acid by prion protein. Biochemistry 46: 1320 –1328, 2007. 3. Boo YC. Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells. Exp Mol Med 38: 63–71, 2006. 4. Broemer M, Krappmann D, Scheidereit C. Requirement of Hsp90 activity for I␬B kinase (IKK) biosynthesis and for constitutive and inducible IKK and NF-␬B activation. Oncogene 23: 5378 –5386, 2004. 5. Chen B, Piel WH, Gui L, Bruford E, Monteiro A. The HSP90 family of genes in the human genome: insights into their divergence and evolution. Genomics 86: 627–637, 2005. 6. Chen G, Cao P, Goeddel DV. TNF-induced recruitment and activation of the IKK complex require Cdc37 and Hsp90. Mol Cell 9: 401–410, 2002. 7. Cortes-Gonzalez C, Barrera-Chimal J, Ibarra-Sanchez M, Gilbert M, Gamba G, Zentella A, Flores ME, Bobadilla NA. Opposite effect of Hsp90␣ and Hsp90␤ on eNOS ability to produce nitric oxide or superoxide anion in human embryonic kidney cells. Cell Physiol Biochem 26: 657–668, 2010. 8. Field N, Low W, Daniels M, Howell S, Daviet L, Boshoff C, Collins M. KSHV vFLIP binds to IKK-␥ to activate IKK. J Cell Sci 116: 3721–3728, 2003. 9. Fleming I, Busse R. Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol 284: R1–R12, 2003. 10. Fontana J, Fulton D, Chen Y, Fairchild TA, McCabe TJ, Fujita N, Tsuruo T, Sessa WC. Domain mapping studies reveal that the M domain of hsp90 serves as a molecular scaffold to regulate Akt-dependent phosphorylation of endothelial nitric oxide synthase and NO release. Circ Res 90: 866 –873, 2002. 11. Forstermann U, Munzel T. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation 113: 1708 –1714, 2006. 12. Garcia C, Nunez-Anita RE, Thebault S, Arredondo Zamarripa D, Jeziorsky MC, Martinez de la Escalera G, Clapp C. Requirement of phosphorylatable endothelial nitric oxide synthase at Ser-1177 for vasoinhibin-mediated inhibition of endothelial cell migration and proliferation in vitro. Endocrine 45: 263–270, 2014.

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13. Hevel JM, Marletta MA. Nitric-oxide synthase assays. Methods Enzymol 233: 250 –258, 1994. 14. Kim F, Tysseling KA, Rice J, Gallis B, Haji L, Giachelli CM, Raines EW, Corson MA, Schwartz MW. Activation of IKK␤ by glucose is necessary and sufficient to impair insulin signaling and nitric oxide production in endothelial cells. J Mol Cell Cardiol 39: 327–334, 2005. 15. Langer T, Rosmus S, Fasold H. Intracellular localization of the 90 kDa heat shock protein (HSP90␣) determined by expression of a EGFPHSP90␣-fusion protein in unstressed and heat stressed 3T3 cells. Cell Biol Int 27: 47–52, 2003. 16. Lei H, Venkatakrishnan A, Yu S, Kazlauskas A. Protein kinase A-dependent translocation of Hsp90␣ impairs endothelial nitric-oxide synthase activity in high glucose and diabetes. J Biol Chem 282: 9364 –9371, 2007. 17. Li J, Buchner J. Structure, function and regulation of the hsp90 machinery. Biomed J 36: 106 –117, 2013. 18. Lin LY, Lin CY, Ho FM, Liau CS. Up-regulation of the association between heat shock protein 90 and endothelial nitric oxide synthase prevents high glucose-induced apoptosis in human endothelial cells. J Cell Biochem 94: 194 –201, 2005. 19. Martasek P, Liu Q, Liu J, Roman LJ, Gross SS, Sessa WC, Masters BS. Characterization of bovine endothelial nitric oxide synthase expressed in E. coli. Biochem Biophys Res Commun 219: 359 –365, 1996. 20. Miao RQ, Fontana J, Fulton D, Lin MI, Harrison KD, Sessa WC. Dominant-negative Hsp90 reduces VEGF-stimulated nitric oxide release and migration in endothelial cells. Arterioscler Thromb Vasc Biol 28: 105–111, 2008. 21. Millson SH, Truman AW, Racz A, Hu B, Panaretou B, Nuttall J, Mollapour M, Soti C, Piper PW. Expressed as the sole Hsp90 of yeast, the ␣- and ␤-isoforms of human Hsp90 differ with regard to their capacities for activation of certain client proteins, whereas only Hsp90␤ generates sensitivity to the Hsp90 inhibitor radicicol. FEBS J 274: 4453– 4463, 2007. 22. Mohan S, Konopinski R, Yan B, Centonze VE, Natarajan M. High glucose-induced IKK-Hsp-90 interaction contributes to endothelial dysfunction. Am J Physiol Cell Physiol 296: C182–C192, 2009. 23. Mohan S, Koyoma K, Thangasamy A, Nakano H, Glickman RD, Mohan N. Low shear stress preferentially enhances IKK activity through selective sources of ROS for persistent activation of NF-␬B in endothelial cells. Am J Physiol Cell Physiol 292: C362–C371, 2007. 24. Park KA, Byun HS, Won M, Yang KJ, Shin S, Piao L, Kim JM, Yoon WH, Junn E, Park J, Seok JH, Hur GM. Sustained activation of protein kinase C downregulates nuclear factor-␬B signaling by dissociation of IKK-␥ and Hsp90 complex in human colonic epithelial cells. Carcinogenesis 28: 71–80, 2007. 25. Pittet JF, Lee H, Pespeni M, O’Mahony A, Roux J, Welch WJ. Stress-induced inhibition of the NF-␬B signaling pathway results from the insolubilization of the I␬B kinase complex following its dissociation from heat shock protein 90. J Immunol 174: 384 –394, 2005. 26. Roman LJ, Sheta EA, Martasek P, Gross SS, Liu Q, Masters BS. High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli. Proc Natl Acad Sci USA 92: 8428 –8432, 1995. 27. Taherian A, Krone PH, Ovsenek N. A comparison of Hsp90␣ and Hsp90␤ interactions with cochaperones and substrates. Biochem Cell Biol 86: 37–45, 2008. 28. Yang J, Park Y, Zhang H, Xu X, Laine GA, Dellsperger KC, Zhang C. Feed-forward signaling of TNF-␣ and NF-␬B via IKK-␤ pathway contributes to insulin resistance and coronary arteriolar dysfunction in type 2 diabetic mice. Am J Physiol Heart Circ Physiol 296: H1850 –H1858, 2009. 29. Zhang L, Cheng J, Ma Y, Thomas W, Zhang J, Du J. Dual pathways for nuclear factor-␬B activation by angiotensin II in vascular smooth muscle: phosphorylation of p65 by I␬B kinase and ribosomal kinase. Circ Res 97: 975–982, 2005. 30. Zhang M, Vogel HJ. Characterization of the calmodulin-binding domain of rat cerebellar nitric oxide synthase. J Biol Chem 269: 981–985, 1994.

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Corrigendum

Dando R, Pereira E, Kurian M, Barro-Soria R, Chaudhari N, Roper SD. A permeability barrier surrounds taste buds in lingual epithelia. Am J Physiol Cell Physiol 308: C21–C32, 2015. First published September 10, 2014; doi:10.1152/ajpcell.00157.2014 (http://ajpcell.physiology.org/ content/308/1/C21).—The following was inadvertently omitted from the article. The article has been corrected online. NOTE ADDED IN PROOF Holland et al. (Holland VF, Zampighi GA, and Simon SA, Tight junctions in taste buds: possible role in perception of intravascular gustatory stimuli, Chem Senses 16: 69 –79, 1991) reported that ions and certain small molecules penetrated into taste buds in canine lingual epithelium. This could indicate there are species differences in the barrier. Moreover, as we had speculated in our report, Holland et al. (ibid) also suggested that intravascular taste might arise from chemical stimuli reaching receptors located on the apical, chemosensory tips of taste cells.

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