1 Department of Medicine, University of Florida College of Medicine and 2 Research Service, Malcom Randall Veterans Affairs Medical Center, Gainesville, Florida 32608-1197
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ABSTRACT |
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The effects of specific microtubule-active agents on nitric oxide (NO) production were examined in pulmonary artery endothelial cells (PAEC). PAEC were incubated with taxol, which stabilizes microtubules, or nocodazole, which disrupts microtubules, or both for 2-4 h. We then examined NO production, endothelial NO synthase (eNOS) activity, and eNOS association with heat shock protein (HSP) 90. Incubation of PAEC with taxol (15 µM) for 2-4 h resulted in an increase in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. Incubation of PAEC with nocodazole (50 µM) for 2-4 h induced a decrease in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. The presence of taxol in the culture medium prevented the effects of nocodazole on NO production and eNOS activity in PAEC. Geldanamycin, a HSP90 inhibitor, prevented the taxol-induced increase in eNOS activity. Taxol and nocodazole did not affect eNOS, HSP90, and tubulin protein contents in PAEC, as detected using Western blot analysis. These results indicate that the polymerization state of the microtubule cytoskeleton regulates NO production and eNOS activity in PAEC. The changes in eNOS activity induced by modification of microtubules are due, at least in part, to the altered binding of HSP90 to eNOS protein.
lung; endothelium; nitric oxide synthase; heat shock protein 90
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INTRODUCTION |
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PULMONARY ARTERY ENDOTHELIAL CELLS (PAEC) generate nitric oxide (NO) from L-arginine via endothelial NO synthase (eNOS) (21, 22, 31). The synthesis of NO requires NADPH, BH4, flavin adenine dinucleotide, flavin mononucleotide, and O2 as cofactors and results in NO and the coproduct L-citrulline (21, 22, 25, 31). It is known that eNOS is tightly regulated by a variety of transcriptional, posttranscriptional, and posttranslational mechanisms (14, 25). At the posttranslational level, eNOS is thought to be regulated by the interaction between eNOS protein and other proteins, such as caveolin (20), calmodulin (2), bradykinin B2 receptor (13), heat shock protein (HSP) 90 (10, 32), and dynamin (3); by fatty acylation with myristate and palmitate (30); and by phosphorylation (2, 9). Histamine, estrogen, or shear stress promote the association between eNOS and HSP90 and increase the activity of eNOS (10), whereas exposure to hypoxia reduces the association between eNOS and HSP90 and decreases eNOS activity (32). Interactions of eNOS with caveolin or bradykinin B2 receptor lead to inhibition of eNOS activity. The binding of Ca2+/calmodulin to eNOS disrupts this inhibitory eNOS-caveolin or eNOS-bradykinin receptor B2 complex, leading to enzyme activation (14).
Microtubules are composed of dimers of - and
-tubulin as well as
a group of proteins referred to as microtubule-associated proteins
(4). The microtubule system is involved in mitosis, cell
structure, and cell motility. It also determines the cellular localization of membrane organelles such as the endoplasmic reticulum, caveolae, and the Golgi apparatus (4). Tubulin has been
reported to be associated with HSP90 and with calmodulin (4, 8,
27, 28).
Our laboratory has previously reported that eNOS and cationic amino acid transporter (CAT)-1, the major arginine transporter in endothelial cells, exist as a complex within plasmalemmal caveolae of PAEC (19). This specific intracellular localization optimizes NO production by creating directed delivery of substrate (L-arginine) to eNOS. Plasmalemmal caveolae interact with the cytoskeleton through actin microfilaments and microtubules (6, 16). Thus reorganization of the cytoskeleton may influence NO production by affecting the function of caveolar proteins, including eNOS and CAT-1. For example, Cucina et al. (7) found that shear stress induces endothelial cell rearrangement of actin filaments and microtubules with the major axes of the cell, and Hutcheson and Griffith (11) reported that shear stress-induced endothelial cell rearrangement of actin filaments and microtubules provide a transduction pathway between shear stress and NO synthesis. Our laboratory has recently shown that the state of actin microfilaments regulates L-arginine transport and NO production by PAEC (33). Therefore, reorganization of the cytoskeleton, including actin filaments and microtubules, may affect eNOS activity, leading to the alteration of NO production.
In the present study, we examined the effect of specific microtubule-active agents on NO production and eNOS activity in PAEC. We found that the state of microtubule polymerization regulates NO production by modulating the binding of HSP90 to eNOS.
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MATERIALS AND METHODS |
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Materials. Taxol, nocodazole, geldanamycin, and anti-tubulin antibody were obtained from Sigma-Aldrich (St. Louis, MO). L-[3H]arginine was obtained from Amersham (Arlington Heights, IL). Mouse anti-eNOS monoclonal antibody and mouse anti-HSP90 monoclonal antibody were obtained from Transduction Laboratories (Lexington, KY). Fluorescence probe 4,5-diaminofluorescein diacetate (DAF-2) used for NO detection was purchased from Calbiochem (San Diego, CA).
Cell culture. Endothelial cells were obtained from the main pulmonary artery of 6- to 7-mo-old pigs and were cultured as previously reported (32). Third- to sixth-passage cells in monolayer culture were maintained in RPMI 1640 medium containing 4% fetal bovine serum and antibiotics (10 U/ml penicillin, 100 µg/ml streptomycin, 20 µg/ml gentamycin, and 2 µg/ml Fungizone) and were used 2 or 3 days after confluence.
All monolayers were initially identified as endothelial cells by phase-contrast microscopy. Selected dishes of cells were further characterized by electron microscopy or by indirect immunofluorescent staining for factor VIII antigen or both. By use of these techniques, monolayer cultures were estimated to be pure endothelial cells.Measurement of intracellular NO production. NO production by intact PAEC in this study was measured by two independent methods. In the first method, intracellular NO production in control cells and cells treated with microtubule-active agents was determined using the NO indicator dye DAF-2 as recently described (24, 33). The cells were loaded with the membrane-permeable diacetate form of DAF-2, which is cleaved by cellular esterases to a membrane-impermeable form. This dye is then capable of combining with intracellularly generated NO to yield a brightly fluorescent triazolofluorescein derivative. The cells were incubated with 10 µM DAF-2 for 60 min at 37°C. After being washed, the cells were treated with 15 µM taxol and 50 µM nocodazole. The fluorescent values were monitored in real time by a BIO-TEK microplate fluorescence reader for 4 h. In some experiments, NG-nitro-L-arginine methyl ester (L-NAME), a specific eNOS inhibitor, was used to inhibit NO production. Our results indicate that incubation of PAEC with L-NAME resulted in a 90% inhibition of NO production, suggesting that this method is reliable to detect the alteration of NO production in PAEC.
In the second method of measuring NO production, intracellular L-citrulline production was determined by monitoring the conversion of L-[3H]arginine into L-[3H]citrulline as previously described (33). Control cells and cells incubated for 4 h with 15 µM taxol or 50 µM nocodazole or both were washed once in 1 ml of warmed LiCl-Dulbecco's modified Eagle's medium (DMEM) and then incubated in 0.5 ml of LiCl-DMEM containing L-[3H]arginine (5 µCi/ml) for 15 min. After the 15-min incubation, PAEC were washed three times with 2 ml of ice-cold LiCl-DMEM containing 5 mM EDTA and were then lysed in 1 ml of 10 mM HCl containing 0.1% SDS. Two aliquots of lysates (100 µl each) were removed for measurements of protein content by the method of Lowry et al. (17). To the remaining samples (0.9 ml), 0.1 ml of 0.2 M sodium acetate buffer, pH 13.0, containing 10 mM L-citrulline was added. The samples were then applied to a column of Dowex AG50WX 8 (H+ form). The effluents, which contained L-[3H]citrulline, were collected in scintillation vials and subjected to scintillation spectrometry. The results are expressed as percentage of control.Determination of eNOS activity.
After incubation with taxol or nocodazole, PAEC monolayers were scraped
and homogenized in buffer A (50 mM Tris · HCl, pH 7.4, containing 0.1 mM each of EDTA and EGTA, 1 mM phenylmethylsulfonyl fluoride, 1.0 µg/ml leupeptin, and 10 µM calpain inhibitor-1). The
homogenates were centrifuged at 100,000 g for 60 min at
4°C, and the total membrane pellets were resuspended in buffer
B (buffer A + 2.5 mM CaCl2). The
resulting suspensions were used for determination of eNOS activity by
monitoring the formation of L-[3H] citrulline
from L-[3H]arginine (32). The
total membrane fractions (100-200 µg protein) were incubated
(total vol 0.4 ml) in buffer B containing 1 mM NADPH, 100 nM
calmodulin, 10 µM BH4, and 5 µM combined
L-arginine and purified
L-[3H]arginine (0.6 µCi; specific activity
69 Ci/mmol; NEN, Boston, MA) for 30 min at 37°C. Purification of
L-[3H]arginine and measurement of
L-[3H]citrulline formation were carried out
as described previously (32). The specific activity of NOS
is expressed as L-citrulline pmol · min1 · mg
1 of
protein and reflects eNOS activity since our cells do not exhibit basal
or taxol-induced inducible NOS activity. Protein contents in the total
membrane fractions were determined by the method of Lowry et al.
(17).
Western blot analysis of eNOS protein, HSP90, and tubulin. After exposure to taxol or nocodazole, PAEC were washed with PBS and then lysed in boiling sample buffer (0.06 M Tris · HCl, 2% SDS, and 5% glycerol, pH 6.8). The lysates were boiled in a water bath for 5 min and were centrifuged to remove insoluble materials. The lysate proteins (20 µg) were fractionated on 7.5% SDS-PAGE gels and blotted onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). The blots were incubated in blocking solution (10 mM Tris · HCl, 0.2% nonfat milk, 100 mM NaCl, 0.1% Tween 20, pH 7.5) and then hybridized with 1:5,000 diluted monoclonal antibodies against eNOS or HSP90 or alkaline phosphatase-labeled anti-tubulin antibody at room temperature for 1 h. The bands were detected using an immunochemiluminescence method, and the density of the bands was quantitated using a Fluor-S-MultiImager (Bio-Rad).
Coimmunoprecipitation. Control PAEC and PAEC exposed to taxol (15 µM) or nocodazole (50 µM) were lysed in ice-cold buffer containing 20 mM Tris · HCl, pH 7.4, 2.5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 100 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 1 mM Pefabloc, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 10 µM calpain inhibitor-1, and 1 µg/ml pepstatin A. The cell lysates were centrifuged at 10,000 g for 20 min to remove insoluble material. Lysates (500 µg, 500 µl) from control and taxol- or nocodazole-treated cells were incubated with 2.5 µg of anti-eNOS antibody, nonimmune IgG, or anti-tubulin antibody at 4°C overnight. Protein A-Sepharose (30 µl) was added, and the samples were further incubated for 2 h at 4°C. The immunoprecipitates were recovered by centrifugation and washed three times in buffer containing 50 mM Tris · HCl, pH 7.5, 150 mM NaCl, and 0.1% Triton X-100. Immunoprecipitated proteins were eluted from the Sepharose beads by boiling the samples for 5 min in 30 µl of SDS immune-blotting sample buffer. The Sepharose beads were then pelleted by centrifugation at 10,000 g, and eNOS, HSP90, or tubulin protein contents in the supernatants were analyzed by Western blot as described above.
Statistical analysis. In each experiment, experimental and control endothelial cells were matched for cell line, age, seeding density, number of passages, and number of days postconfluence to avoid variation in tissue culture factors that can influence the measurement of eNOS activity and eNOS protein analysis. Results are shown as means ± SE for n experiments. Student's paired t-test was used to determine the significance of differences between the means of treated and control groups, and a P value of <0.05 was taken as significant.
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RESULTS |
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Modification of microtubule cytoskeleton.
To define the effects of microtubule stabilization or disruption on NO
production and eNOS, we used taxol, a specific microtubule stabilizer
(29), and nocodazole, which inhibits microtubule polymerization (15). Incubation of PAEC with 15 µM taxol
for 2-4 h resulted in significantly more microtubule
polymerization, and incubation of PAEC with nocodazole (1-50 µM)
for 2-4 h induced disruption of microtubules (Fig.
1). As shown in Fig. 1, the
effect of taxol (15 µM) on microtubule polymerization in PAEC was
prevented by nocodazole (50 µM). With the concentrations mentioned
above, taxol and nocodazole did not cause cell damage, as detected by trypan blue exclusion and lactate dehydrogenase release.
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Effect of taxol and nocodazole on intracellular NO and
L-citrulline production in intact PAEC.
To confirm that microtubule reorganization regulates NO production,
PAEC monolayers were incubated with taxol (15 µM), nocodazole (50 µM), or both for 2-4 h. NO and L-citrulline
production were then determined. As shown in Fig.
2, incubation of PAEC with taxol resulted
in an increase in NO production, and incubation with nocodazole
resulted in a decrease in NO production, measured with DAF-2
fluorescent dye. The effect of taxol on NO production in PAEC was
prevented by nocodazole, suggesting that the changes in NO production
are induced by the modification of microtubules. The taxol- and
nocodazole-induced changes in NO measured by intracellular L-citrulline content were very similar to those observed
with DAF-2, thus confirming the reliability of the NO fluorescent assay (Fig. 3).
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Effect of taxol and nocodazole on eNOS activity of total membrane
fractions from PAEC.
To investigate the effect of microtubule reorganization on eNOS
activity, PAEC were incubated with taxol (15 µM) or nocodazole (50 µM) for 2-4 h. As shown in Fig. 4,
incubation of PAEC with taxol resulted in an increase in eNOS activity
in the total membrane fraction from PAEC. The taxol-induced increase in
eNOS activity was reversible on removal of taxol from the medium.
Nocodazole prevented the effect of taxol on eNOS activity. Incubation
of PAEC with nocodazole caused a decrease in eNOS activity in a dose- and time-dependent manner (Fig. 5).
Incubation of PAEC with nocodazole-free culture medium for 24 h
after exposure to nocodazole (50 µM) for 4 h resulted in a
recovery of eNOS activity.
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Coimmunoprecipitation of eNOS, HSP90, and tubulin.
Research has documented that HSP90 forms a complex with eNOS and
activates eNOS activity (10, 32). Moreover, several
reports have shown that HSP90 is colocalized to microtubules (4,
8, 27, 28). Therefore, it is possible that microtubules may be associated with the HSP90-eNOS complexes in PAEC and that HSP90 may
mediate the effect of microtubule reorganization on eNOS activity. To
test these possibilities, the lysates from PAEC exposed to taxol or
nocodazole were immunoprecipitated with antibody directed against eNOS
or tubulin. Immunoprecipitation of eNOS from PAEC lysates resulted in
the coprecipitation of HSP90. Treatment of PAEC with taxol
significantly increased the amount of HSP90 coimmunoprecipitated with
eNOS protein (Fig. 6). In contrast,
treatment of PAEC with nocodazole decreased the amount of HSP90
coimmunoprecipitated with eNOS (Fig. 7).
Immunoprecipitation of tubulin from PAEC lysates resulted in the
coprecipitation of eNOS and HSP90. However, treatment of PAEC with
taxol or nocodazole did not alter the ratio of eNOS to tubulin that is
immunoprecipitated by anti-tubulin antibody or the ratio of HSP90 to
tubulin that is immunoprecipitated by anti-tubulin antibody (Fig.
8). To investigate the possibility that
microtubule reorganization might influence the protein contents of
tubulin, eNOS, and HSP90, we measured the protein contents of tubulin,
eNOS, and HSP90 in control and taxol- or nocodazole-treated cells using
Western blot analysis. Exposure of PAEC to 15 µM taxol or 50 µM
nocodazole for 2-4 h did not induce significant changes in
tubulin, eNOS, or HSP90 protein contents (Fig.
9).
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Effect of geldanamycin on taxol-induced activation of eNOS
activity.
To evaluate further the role of HSP90 in the microtubular regulation of
eNOS activity, we used geldanamycin, a specific inhibitor of HSP90.
Geldanamycin binds to the nucleotide-binding site of HSP90 and
specifically blocks HSP90 function. Incubation of PAEC with
geldanamycin for 4 h prevented the taxol-induced increase in eNOS
activity (Fig. 10). Geldanamycin did
not inhibit eNOS activity when added directly to the total membrane
fraction of PAEC.
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DISCUSSION |
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In the present study, we have shown that pharmacological modification of microtubule organization alters NO production by PAEC. Stabilization of microtubules by taxol resulted in an increase in NO production, whereas disruption of microtubules by nocodazole decreased NO production. Our results indicate that the inhibitory effect of nocodazole on NO production can be prevented by taxol, suggesting that the alteration of NO production induced by taxol and nocodazole is related to modification of microtubule polymerization state.
Our results also indicate that stabilization of microtubules by taxol increases eNOS activity in PAEC, whereas disruption of microtubules by nocodazole results in a decrease in eNOS activity without significant alteration of eNOS protein content. To explore the mechanism by which microtubules regulate eNOS activity, we first considered the possibility that alteration in the state of tubulin polymerization may influence eNOS activity through a tubulin-eNOS interaction. Our results indicate that eNOS protein can be coimmunoprecipitated by anti-tubulin antibody, suggesting that eNOS is associated with tubulin. However, neither taxol nor nocodazole influenced the ratio of eNOS to tubulin that is immunoprecipitated by anti-tubulin antibody, indicating that modifications of tubulin polymerization do not influence eNOS-tubulin association. Therefore, it seems unlikely that microtubule polymerization influences eNOS activity directly.
Calmodulin has been reported to interact with both microtubules and eNOS and therefore might be responsible for the alteration of eNOS activity caused by microtubule reorganization. Pirollet et al. (26) reported that calmodulin is associated with microtubules in bovine brain. However, calmodulin is unlikely to be responsible for the microtubular regulation of eNOS activity in PAEC because 100 nM calmodulin was included in the reaction mixture used to measure eNOS activity in our in vitro assay.
HSP90, a chaperone protein, also interacts with eNOS and microtubules. Sanchez et al. (28), using coimmunoabsorption and coimmunofluorescence techniques, found that HSP90 is associated with tubulin in fibroblasts and Ptk (rat kangaroo kidney) cells. Czar et al. (8) also reported that HSP90 colocalized to microtubules in rat pulmonary endothelial cells. Consistent with these observations, we found that HSP90 coimmunoprecipitates with tubulin, indicating HSP90 is associated with tubulin. However, neither taxol nor nocodazole affected the ratio of HSP90 to tubulin that is immunoprecipitated by anti-tubulin antibody, suggesting that modifications of microtubule polymerization do not influence HSP90-tubulin interaction. Recently, Garcia-Cardena et al. (10) and we (32) reported that binding of HSP90 to eNOS enhances the activation of eNOS. The present study indicates that the HSP90 protein content that was coimmunoprecipitated by the anti-eNOS antibody increased in the taxol-treated PAEC, whereas the HSP90 protein content that was coimmunoprecipitated by anti-eNOS antibody decreased in the nocodazole-treated cells, suggesting that HSP90 may be responsible for the alteration of eNOS activity caused by microtubule reorganization. Moreover, the specific inhibitor of HSP90, geldanamycin, prevented the taxol-induced increase in eNOS activity in PAEC. Correspondingly, geldanamycin also prevented the taxol-induced increase in HSP90 protein content that was coimmunoprecipitated by the anti-eNOS antibody in PAEC (data not shown). Together, these results indicate that an eNOS-HSP90-tubulin complex exists in PAEC. Modifications of tubulin polymerization do not influence eNOS-tubulin interactions or HSP90-tubulin interactions but do affect eNOS-HSP90 interactions. Increased tubulin polymerization may bring HSP90 closer to eNOS, thus promoting the association between HSP90 and eNOS that leads to enhanced eNOS activity. Tubulin depolymerization, on the other hand, decreases the association between HSP90 and eNOS. Thus increased association of eNOS with HSP90 is responsible for increased eNOS activity and NO production induced by pharmacological stabilization of microtubules, and decreased association of eNOS with HSP90 is responsible for decreased eNOS activity and NO production induced by pharmacological disruption of microtubules.
Regulation of eNOS function by microtubules is a novel concept that advances our understanding of the regulation of NO production in the pathophysiology of hypoxia, pulmonary hypertension, and angiogenesis. For example, hypoxia induces microtubule disruption (12), and it also decreases eNOS activity and NO production in cultured PAEC (1, 32). Increases in shear stress in pulmonary hypertension also change cytoskeletal organization (7, 18), and several studies have reported reduced eNOS activity and/or NO production in the presence of pulmonary hypertension (1, 5). Recent reports indicate that NO is an important mediator of angiogenesis that is regulated by microtubules (23), suggesting that microtubule regulation of eNOS activity modulates angiogenesis. Moreover, vascular endothelial growth factor has also been shown to stimulate the recruitment of HSP90 to eNOS (10), suggesting that HSP90-eNOS association might be common to multiple pathways that lead to angiogenesis in the pulmonary vasculature. Finally, demonstration of microtubule regulation of eNOS opens the door to the possibility that manipulation of the microtubule system may provide a new avenue for preventing or reversing impaired eNOS activity and vascular NO production in the presence of endothelial dysfunction.
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ACKNOWLEDGEMENTS |
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We thank Humberto Herrera for assistance with tissue culture, Dr. Sophia Edward-Bennett for help with immunoprecipitation, Dr. Michael Bubb for critical review, and Janet Wootten for editorial assistance.
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FOOTNOTES |
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This work was supported by the Medical Research Service of the Department of Veterans Affairs, National Heart, Lung, and Blood Institute Grant HL-52136, and American Heart Association Florida Affiliate Grant 0130450B.
Address for reprint requests and other correspondence: Y. Su, Research Service (151), VA Medical Center, 1601 S.W. Archer Road, Gainesville, FL 32608-1197 (E-mail: ysu{at}ufl.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 4, 2002;10.1152/ajplung.00388.2001
Received 2 October 2001; accepted in final form 20 December 2001.
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