Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
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Disruption of microfilaments in human umbilical vein endothelial cells (HUVEC) with cytochalasin D (cytD) or latrunculin A (latA) resulted in a 3.3- to 5.7-fold increase in total synthesis of prostaglandin E2 (PGE2) and a 3.4- to 6.5-fold increase in prostacyclin (PGI2) compared with control cells. Disruption of the microtubule network with nocodazole or colchicine increased synthesis of PGE2 1.7- to 1.9-fold and PGI2 1.9- to 2.0-fold compared with control cells. Interestingly, however, increased release of PGE2 and PGI2 from HUVEC into the media occurred only when microfilaments were disrupted. CytD treatment resulted in 6.7-fold more PGE2 and 3.8-fold more PGI2 released from HUVEC compared with control cells; latA treatment resulted in 17.7-fold more PGE2 and 11.2-fold more PGI2 released compared with control cells. Both increased synthesis and release of prostaglandins in response to all drug treatments were completely inhibited by NS-398, a specific inhibitor of cyclooxygenase-2 (COX-2). Disruption of either microfilaments using cytD or latA or of microtubules using nocodazole or colchicine resulted in a significant increase in COX-2 protein levels, suggesting that the increased synthesis of prostaglandins in response to drug treatments may result from increased activity of COX-2. These results, together with studies demonstrating a vasoprotective role for prostaglandins, suggest that the cytoskeleton plays an important role in maintenance of endothelial barrier function by regulating prostaglandin synthesis and release from HUVEC.
cyclooxygenase; microfilaments; microtubules; vasoprotection; prostaglandin E2; prostacyclin
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INTRODUCTION |
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THE ACTIN CYTOSKELETON PLAYS a complex role in the maintenance of endothelial barrier function. Tight cell-cell adhesion and strong attachments to the extracellular matrix through focal adhesions correlates with low endothelial permeability (4, 14, 19, 35). Many agonists, like thrombin, increase axial contractility by inducing actin-myosin stress fiber formation that promotes formation of intracellular gaps and compromises barrier function (14, 15). Additionally, disassembly of actin filaments using pharmacological agents also decreases endothelial integrity and barrier function (3, 27, 33, 37).
Several studies have demonstrated that cytoskeletal reorganization and loss of barrier function correlate with increased release of prostaglandin E2 (PGE2) and prostacyclin (PGI2) (11, 25, 28, 39). Important steps in the regulation of prostaglandin synthesis include cleavage of arachidonic acid (AA) from membrane phospholipids by phospholipase A2 (PLA2) and the conversion of AA to prostaglandin H2 by cyclooxygenases (COX) (20, 21). Activity of the two isoenzymes of COX, cyclooxygenase-1 (COX-1), which is constitutively expressed, and cyclooxygenase-2 (COX-2), which is induced in many types of cells by a variety of agonists, (9, 10) is controlled primarily at the level of transcription and translation. However, some posttranslational modification of the enzyme also occurs that may regulate activity (32). The mechanisms that regulate prostaglandin production by cells, however, remain largely unknown. Also unclear is whether prostaglandin release from cells occurs simply as a result of increased prostaglandin synthesis or if prostaglandin release is an actively regulated process that can be controlled independently of increased synthesis.
PGE2 and PGI2 are vasodilatory prostaglandins released by endothelial cells (34). Release of PGI2 from endothelial cells is stimulated by many factors, including hypoxia, lipopolysaccharide (LPS), endotoxin, and fluid shear stress (1, 18, 23, 30). Each of these stimuli also increases production of COX-2 mRNA and protein by endothelial cells (1, 8, 22, 23, 30). Under conditions in which endothelial barrier function is compromised, PGE2 and PGI2 have been shown to play a vasoprotective role. That is, these prostaglandins can protect the vasculature from damage associated with conditions in which barrier permeability is increased (6, 24). Welles et al. (43) suggested that PGI2 could actually decrease endothelial permeability in the microvasculature by promoting the reorganization of microfilaments. Thus PGE2 and PGI2 release may be coordinately regulated, at least in part, by changes in the normal organization of cytoskeletal filaments as part of a vasoprotective response that helps maintain the integrity of endothelial barrier function in the vasculature.
In this study, we have examined directly the effect of microfilament and microtubule disruption on PGE2 and PGI2 synthesis and release in human umbilical vein endothelial cells (HUVEC). Disruption of either microtubules (using nocodazole or colchicine) or microfilaments [using cytochalasin D (cytD) or latrunculin A (latA)] resulted in significant increases in prostaglandin synthesis, whereas only disruption of the actin cytoskeleton increased release of PGE2 and PGI2 from HUVEC. These studies demonstrate that increased synthesis of prostaglandins in HUVEC does not always result in increased prostaglandin release and suggest that actin filament-regulated PGE2 and PGI2 release may represent a vasoprotective response during conditions in which endothelial barrier function is compromised.
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MATERIALS AND METHODS |
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Cell culture. HUVEC were obtained from Clonetics (East Rutherford, NJ) and grown in endothelial growth media (Clonetics) containing 2% fetal bovine serum, 12 µg/ml bovine brain extract, 1 µg/ml human epidermal growth factor, and 1 µg/ml hydrocortisone, gentamicin, and amphotericin B. Cells were maintained in 5% CO2 at 37°C, and experiments were performed using cells between passages 2 and 4.
Experimental protocol. Cells were grown on glass slides coated with 10 µg/ml of human plasma fibronectin (GIBCO Life Technologies, Carlsbad, CA) until confluent. Cells were pretreated for 1 h with either vehicle control [0.1% dimethyl sulfoxide (DMSO)], 0.2 µM cytD, 50 nM latA, 10 µM nocodazole, or 10 µM colchicine or overnight with 10 µM NS-398. DMSO, cytD, nocodazole, and colchicine were purchased from Sigma Chemical (St. Louis, MO); latA and NS-398 were purchased from Calbiochem (La Jolla, CA). The cells were incubated for 6 h in DMEM containing 0.5% FCS and the appropriate pharmacological agent. At the end of the 6-h treatment, the cells were collected for either immunocytochemistry, Western blot analysis, or prostaglandin measurement.
Immunocytochemistry.
Cells were rinsed twice in phosphate-buffered saline (PBS) and
preserved in 4% paraformaldehyde for 15 min. Microtubules were visualized by staining with an anti--tubulin antibody (Sigma) followed by an appropriate rhodamine-labeled secondary antibody (Jackson Immunoresearch, West Grove, PA). Microfilaments were stained
with FITC-phalloidin (Molecular Probes, Eugene, OR). Images were
recorded using an RT Color Spotdigital camera (Diagnostic Instruments,
Sterling Heights, MI) using an Optiphot-2 Nikon epifluorescent microscope.
Western blot analysis. Cells were harvested by direct lysis in SDS sample buffer and homogenized by shearing through a 25-gauge needle. Protein concentrations were determined using the amido black method (38). Equal protein (20 µg) was loaded onto a 7.5% SDS-PAGE gel for separation and transferred to nitrocellulose for immunoblot analysis. COX-1 and COX-2 antibodies were purchased from Cayman; the vinculin antibody, VIN-11-5, was obtained from Sigma Chemical. The appropriate horseradish peroxidase-labeled secondary antibodies were obtained from Jackson Immunoresearch, and the antibody signal was detected by enhanced chemiluminescence. Each experiment was carried out in at least triplicate, and scanning densitometry of bands was performed with the Bio-Rad (Hercules, CA) Molecular Analyst program.
Prostaglandin measurement. Prostaglandin release from cells was measured by incubating the cells in 1 ml of DMEM containing the appropriate drugs for 30 min after the 6-h treatment. The media were collected and centrifuged at 12,000 g for 2 min to pellet any particulates. The supernatant was transferred to a fresh tube and retained for prostaglandin analysis. To determine the amount of prostaglandins retained in the cells after drug treatment, cells were rinsed twice with PBS, harvested in a hypotonic buffer (50 mM Tris, 0.1 mM EGTA, pH 7.4), and incubated for 5 min on ice before removal of the insoluble material by centrifugation at 12,000 g for 2 min. The supernatant was transferred to a fresh tube and retained for prostaglandin analysis. PGE2 and PGI2 were measured with the appropriate enzyme immunoassay kit from Amersham Pharmacia Biotech (Piscataway, NJ) according to the manufacturer's instructions. Results were normalized to total cellular protein as determined by the bicinchoninic acid reagent (BCA; Pierce Chemical, Rockford, IL) in the lysates.
PLA2 activity assays. PLA2 was measured using an activity kit purchased from Cayman Chemical (Ann Arbor, MI) according to the manufacturer's instructions.
Cell viability assay and apoptosis detection.
Cell viability was determined by trypan blue exclusion. After
treatment with the appropriate pharmacological agent, cells were
harvested by trypsinization and incubated in 0.04% trypan blue (Sigma
Chemical) for 4 min. Cells were counted with a hemacytometer, and the
number of cells retaining the dye (nonviable) and the number of
unstained cells was noted. To determine whether drug treatments induced
apoptosis in HUVEC, DNA strand breaks were detected using a
TdT-mediated dUTP nick end labeling (TUNEL) assay (Boehringer Mannheim,
Indianapolis, IN). Cells were treated with the appropriate
pharmacological agent for 6 h and then preserved in 4%
paraformaldehyde. Cells were stained by TUNEL reaction according to the
manufacturer's instructions. As a positive control, HUVEC were treated
with 10 ng/ml of human tumor necrosis factor (TNF)- (Sigma Chemical)
for 24 h and were stained with the TUNEL antibody. At least 50 cells were counted for each treatment, and the percentage of cells that
were apoptotic was determined.
Immunoprecipitation. After treatment, cells were harvested in immunoprecipitate buffer (10 mM Tris, pH 7.4, 145 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM vanadate, and 1 mM NaFl with phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and aprotinin added as needed) and incubated on ice for 5 min before removing the insoluble material by centrifugation (5 min, 14,000 rpm). Protein concentration was determined by the BCA assay, and equal amounts of protein from each sample were incubated with 5 µg of COX-2 antibody for 1 h at 4°C. The lysates were then incubated with 100 µl of 10% rabbit anti-mouse protein A-Sepharose beads for 1 h. The beads were washed five times with immunoprecipitation buffer before extraction in SDS sample buffer and Western blot analysis.
Statistical analysis. Statistical analysis of the scanning densitometry of COX-1 and COX-2 and the PGE2 and PGI2 measurements was made using the statistical package Statview.
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RESULTS |
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Effects of cytoskeletal inhibitors on HUVEC.
HUVEC were treated for 6 h with either cytD (0.2 µM) or latA
(0.05 µM) to disrupt microfilaments or with nocodazole (10 µM) or
colchicine (10 µM) to disrupt microtubules. These drug concentrations were chosen based on the ability to effectively disrupt the
appropriate cytoskeletal network (Fig. 1)
without decreasing cell viability (assessed by trypan blue exclusion)
or inducing apoptosis (based on TUNEL staining). Cell viability
in all conditions was never less than that of the 0.1% DMSO control
treatment (88% viable after trypsinization). In cells treated with
TNF-, 18.5% of the cells stained positive by the TUNEL assay,
whereas only 1.3% of the vehicle-treated cells stained positive. The
percentage of apoptotic cells measured by positive TUNEL staining
was 2.2% in both the cytD- and latA-treated cells and 1.2 and 1.5% in
the nocodazole- and colchicine-treated cells, respectively.
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Effects of cytoskeletal disruption on prostaglandin synthesis and
release.
To directly examine the effect of cytoskeletal disruption on
prostaglandin synthesis in HUVEC, cells were treated with cytD, latA,
nocodazole, colchicine, or vehicle (0.1% DMSO) alone for 6 h and
then incubated for 30 min in 1 ml of medium. PGI2 and PGE2 in the media and in cell lysates were measured by
enzyme immunoassay. All four drugs significantly increased total
PGE2 and PGI2 synthesis, defined as the amount
of each prostaglandin released into the media plus the amount retained
in cell lysates (Fig. 2).
PGE2 and PGI2 synthesis in HUVEC treated with
cytD was 3.3- and 3.4-fold higher compared with control cells
(P < 0.002; Table 1);
HUVEC treated with latA, PGE2, and PGI2 were
5.7- and 6.6-fold higher, respectively, compared with control cells
(P < 0.003; Table 1). Nocodazole-treated HUVEC
produced 1.7-fold more PGE2 and 2.0-fold more
PGI2 than control cells (P < 0.02 PGE2, P < 0.0004 PGI2; Table
1); colchicine-treated HUVEC produced 1.9-fold more PGE2
and 1.9-fold more PGI2 than control cells
(P < 0.004; Table 1). The increased levels of
PGI2 and PGE2 in HUVEC treated with any
of these drugs were completely inhibited by NS-398, a specific
inhibitor of the enzyme COX-2. NS-398 also significantly inhibited
synthesis of PGE2 from controls cells (P < 0.01) and inhibited basal levels of PGI2 (P < 0.08), suggesting that in the absence of cytoskeletal disruption,
basal PGE2 and PGI2 synthesis in HUVEC is
dependent on COX-2 activity.
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Effects of cytoskeletal disruption on PLA2 activity and
COX-2 tyrosine phosphorylation.
To investigate the potential role of cytoskeleton-sensitive changes in
PLA2 activity in increased prostaglandin production, PLA2 activity assays were performed on cell extracts after
disruption of either microfilaments or microtubules. However, all drug
treatments significantly decreased PLA2 activity compared
with control activity (P < 0.01; Fig.
5). Thus cytoskeletal disruption does not
increase prostaglandin production through an effect on
PLA2.
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Effect of cytoskeletal disruption on COX-2 protein levels.
To determine whether the increased synthesis of PGE2 and
PGI2 in cytD-treated cells might result from increased
expression of COX-2, the inducible isoform of COX, immunoblot analysis
of protein extracts, was performed (Fig.
7). Densitometry of a minimum of four
separate experiments (summarized in Table
2) indicated a statistically significant
increase in COX-2 protein levels in cells treated with cytD (2.0-fold),
latA (2.4-fold), nocodazole (2.6-fold), or colchicine (2.8-fold),
compared with vehicle-only controls. In contrast, expression of COX-1,
the constitutive form of the enzyme, was not affected in cells treated
with any of these drugs (Fig. 7; Table 2). Thus the increased synthesis
of PGE2 and PGI2 induced by disruption of
either microfilaments or microtubules may be explained, at least in
part, by increased expression of COX-2.
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DISCUSSION |
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Our results support a role for the cytoskeleton in the synthesis and release of prostaglandins from HUVEC. Specifically, disruption of either microfilaments or microtubules resulted in increased synthesis of PGE2 and PGI2 compared with control cells. However, disruption of microfilaments, but not disruption of microtubules, induced a large increase in release of PGE2 and PGI2 into the media compared with control cells. When calculated as a percentage of total prostaglandin synthesis, PGE2 release increased from 21% to 38-51%, and PGI2 release increased from 28% to 39-56% after disruption of the actin cytoskeleton. However, microtubule disruption did not change the percentage of total prostaglandins released compared with control cells. Thus the amount of prostaglandins released from cells is not always proportional to total increases in prostaglandin synthesis. Instead, prostaglandin release may be actively regulated by the actin cytoskeleton. It is not clear why treatment of cells with latA results in significantly higher PGE2 and PGI2 production and release compared with cells treated with cytD. The explanation for this difference might lie in the fact that although both drugs lead to complete disassembly of actin stress fibers, the mechanisms of action of these two drugs are very different. While latA sequesters actin monomers and prevents their incorporation into filaments, cytD severs actin filaments and caps their barbed ends. Others (13, 42) have reported that cytD treatment results in much higher levels of insoluble F-actin remaining in cells compared with cells treated with latA. The increased synthesis of both PGE2 and PGI2 by cells with a disrupted actin or microtubule cytoskeleton was inhibited by NS-398 and was thus COX-2 dependent. Microfilament or microtubule disruption did not increase PLA2 activity in cell extracts using an in vitro PLA2 activity. However, COX-2 protein levels were increased significantly, suggesting that both filament systems play a role in the regulation of COX-2 protein expression and activity in HUVEC. On the basis of the data presented in this study, we conclude that COX-2 protein levels in cells and synthesis of prostaglandins is regulated by both microfilaments and microtubules, while release of prostaglandins is modulated, at least in part, by the actin cytoskeleton.
Others (12) have shown that endothelial cells with altered cytoskeletal organization increased release of PGI2; however, these authors altered the actin cytoskeleton by plating the cells on substrates with differing adhesive capacities. Cells that were poorly adhered had a reduced actin network and released more PGI2. It is possible, however, that in those experiments, altering the adhesive extracellular matrix alone may also influence prostaglandin release. In our experiments, microfilaments and microtubules were directly disrupted using pharmacological agents at concentrations that did not compromise cell viability or induce apoptosis and had minimal affect on the morphology of the cell (Fig. 1). Thus our experiments utilize multiple cytoskeletal poisons that function through different mechanisms to demonstrate a direct relationship between cytoskeletal organization and synthesis and release of PGE2 and PGI2.
In other cell types, including human mammary epithelial cells (41) and rat osteoblasts (45), disruption of microtubules increased release of PGE2. In contrast, our studies with HUVEC show that microtubule disruption using nocodazole or colchicine increased PGE2 and PGI2 synthesis but did not increase prostaglandin release. In agreement with our results using HUVEC, treatment of epithelial cells with cytD or nocodazole increased COX-2 protein expression (41). When HUVEC were treated with nocodazole or colchicine to disrupt microtubules, we observed an apparent increase in the number and thickness of stress fiber bundles. Increased stress fiber formation has been observed previously in fibroblasts in response to microtubule disruption (7). The lack of prostaglandin release from HUVEC treated with nocodazole or colchicine, however, suggests that some other signal is required for release of the prostaglandins into the extracellular environment.
Disruption of the actin or microtubule cytoskeleton could increase prostaglandin synthesis by increasing COX-2 activity through a mechanism involving posttranslational modification of COX-2 by tyrosine phosphorylation, as previously suggested (32). However, we found no evidence for increases in tyrosine phosphorylation of COX-2 in response to disruption of either microfilaments or microtubules (Fig. 6). Alternatively, prostaglandin synthesis could be regulated by the subcellular localization of COX-2. COX-2 is normally localized to the endoplasmic reticulum and nuclear membranes (40). Although immunolocalization of COX-2 in cells with disrupted microfilaments and microtubules did not reveal gross changes in COX-2 localization compared with vehicle-treated controls (Sawyer, unpublished results), it remains possible that accessibility of COX-2 to free AA may be increased by cytoskeletal disruption.
Another possible mechanism for increased prostaglandin synthesis from HUVEC following disruption of microfilaments or microtubules may involve increased membrane fluidity and PLA2 activity. Disruption of microfilaments or microtubules increases membrane fluidity in epithelial cells and leukocytes (26, 31, 44), and increased membrane fluidity activates PLA2 in fibroblast cells (5). Because PLA2 cleaves phospholipids to release AA, the substrate for COX-2 disrupting either microfilaments or microtubules may increase prostaglandin synthesis by activating PLA2 and increasing pools of AA without requiring new COX-2 synthesis. Although after cytoskeletal disruption we measured a decrease in PLA2 in cell extracts using an in vitro PLA2 activity assay, further studies will be required to determine whether changes in membrane fluidity and PLA2 activity might be involved in increased prostaglandin synthesis in HUVEC with a disrupted cytoskeleton.
Both the actin and microtubule cytoskeletons are clearly involved in
the regulation of endothelial barrier function. Treatment of
endothelial cells with TNF- or hypoxia disrupts barrier function and
induces actin depolymerization (16, 17, 29), while LPS disrupts barrier function and depolymerizes microfilaments and microtubules (16). Barrier dysfunction also correlates
with increased release of prostaglandins that are vasoprotective, that is, these prostaglandins may function to protect barrier integrity. For
example, hypoxia, LPS, and endotoxin, all of which cause endothelial barrier dysfunction, also stimulate release of PGI2 and
induce COX-2 mRNA and protein expression (2, 22, 23, 36).
Addition of exogenous prostaglandins, including PGE2 and
PGI2, can actually enhance endothelial barrier function
(24, 43) and prevent hypoxia-induced endothelial barrier
dysfunction (6). Furthermore, prostaglandins that prevent
hypoxia-, LPS-, and endotoxin-induced barrier dysfunction also
stabilize microfilaments. We propose that increased PGE2
and PGI2 released in response to microfilament disruption
may act in a paracrine/autocrine fashion to restore barrier function.
Although the precise mechanisms remain to be determined, our results,
together with studies demonstrating a vasoprotective role for
prostaglandins, suggest an important role for the cytoskeleton in
maintaining endothelial barrier function through regulating
prostaglandin synthesis and release.
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ACKNOWLEDGEMENTS |
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This work was supported by American Heart Association Grant-in-Aid 9750403N and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants AR-45831 and AR-45218 (to F. M. Pavalko). S. J. Sawyer was supported by an American Heart Association-Midwest Affiliate postdoctoral fellowship. S. M. Ponik was supported by a National Aeronautics and Space Administration predoctoral fellowship.
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FOOTNOTES |
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Address for reprint requests and other correspondence: F. M. Pavalko, Dept. of Cellular and Integrative Physiology, 635 Barnhill Dr., Medical Science Bldg., Rm. 2069, Indiana Univ. School of Medicine, Indianapolis, IN 46202 (E-mail: fpavalko{at}iupui.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.
Received 13 November 2000; accepted in final form 2 May 2001.
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