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1Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory, 2Department of Neurology, 3Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia 30322
Submitted 3 April 2003 ; accepted in final form 20 May 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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caveolin-1; mechanosensing; atherosclerosis
The mechanism by which endothelial cells recognize changes in shear stress (mechanosensing) and control activation of multiple signaling pathways in a well-orchestrated manner is not well understood. Several potential mechanosensing systems have been proposed including cytoskeleton/integrins (4, 43), G proteins (14), K+ channels (26), adherens junction proteins (37), and caveolae (27, 28, 30).
Caveolae are noncoated micropatches of the plasma membrane with a variety of shapes (flat, invaginated, and tubular) and are found in most cell types, including endothelial cells, fibroblasts, smooth muscle cells, and adipocytes (1, 25). They carry out at least two different functions: 1) transport of large and small molecules, and 2) transmembrane cell signaling centers (25).
We have shown evidence suggesting that caveolae play an important role in regulation of the ERK pathway by manipulating two of its critical components, plasmalemmal cholesterol and caveolin-1 (27, 28). These studies examined the response of endothelial cells cultured under static conditions to acute shear. Currently, most investigators, including us, in this field use static cultured cells as a physiologically "normal" control. This is necessary due to technical constraints of culturing cells under shear conditions. However, most vascular endothelial cells in vivo are continuously exposed to shear stress and static cultured cells may not represent true "control conditions." Although static cultured cells have been extremely valuable in dissecting endothelial responses to mechanical force, their phenotypes and responses may not represent those occurring under physiological conditions in vivo. For example, Schnitzer and colleagues (36) have suggested that the number of caveolae in endothelial cells decrease during culture compared with their counterparts in vivo.
We hypothesized that caveolae density in the cell surface may be critical in mechanosensing and subsequent mechanosensitive cell signaling. To test this, we examined whether chronic laminar shear exposure changes formation of caveolae, subcellular localization of caveolin-1, and activation of two well-known mechanosensitive signaling proteins (ERK and Akt) in cultured bovine aortic endothelial cells (BAEC). Here, we show that chronic laminar shear stress induces translocation of caveolin-1 from the Golgi to the plasma membrane, increases caveolae formation preferentially at the luminal surface, and changes the activation patterns of ERK and Akt.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Shear stress studies. Cells were exposed to unidirectional laminar shear stress with the use of a parallel-plate shear chamber or a cone-and-plate viscometer as described by us previously (13).
Preparation of RNA or cell lysates. Total cell RNA was extracted from endothelial cells with the use of a RNEasy Mini Kit (Qiagen; Valencia, CA) according to the manufacturer's instructions. cDNA was prepared from total RNA with SuperScript II (Invitrogen; Carlsbad, CA) according to the manufacturer's instructions. For protein analysis, cells were washed in ice-cold PBS, scraped in 0.5 ml of lysis buffer composed of 60 mM octylglucoside, 10 mM Tris (pH 7.4), 150 mM NaCl, 1.0% Triton X-100, 1.0 mM sodium orthovanadate, and 0.1 mM phenylmethylsulfonyl fluoride (28, 45), solubilized for 30 min at 4°C, and centrifuged at 8,000 g for 15 min. The protein content of solubilized lysates was measured with the use of a DC assay kit (Bio-Rad; Hercules, CA) (18).
Northern blot analysis. Total RNA (10 µg) was added to each
lane of a 1% agarose gel then transferred to a nylon membrane (Schleicher and
Schuell; Keene, NH). The RNA was UV cross-linked to the membrane with the use
of Stratalinker (Stragene; La Jolla, CA). The blot was prehybridized with 1 M
NaCl, 5x Denhardt's solution, 50 mM Tris, pH 7.4, 50% Formamide, 0.5%
SDS (wt/vol), and 0.01% salmon sperm DNA (vol/vol) for 2 h at 42°C in a
hybridization oven (Thermo Hybrid; Franklin, MA) before an overnight
hybridization (prehybridization solution containing 50 µCi
[
-32P]dCTP but without Denhardt's solution) and probed with
cDNA specific for caveolin-1 (5'-GTATTTGCCCCCAGACATGCTGGC-3')
(32). The blots were affixed
to a Phosphor screen (Molecular Dynamics; Sunnyvale, CA) and scanned on an
autoradiography system (Storm; Molecular Dynamics). Densiometric
quantification was performed with the use of Image software (Scion; Frederick,
MD) and normalized to 18S rRNA.
Western blotting. Total lysates (20 µg/well) were resolved by 10% SDS-PAGE, transferred to a polyvinylidine difluoride membrane (Millipore; Bedford, MA), and probed with antibodies specific to caveolin-1, endothelial nitric oxide synthase (eNOS; Transduction Laboratories, Lexington; KY) and actin (Santa Cruz Biotechnology; Santa Cruz, CA). Goat anti-rabbit or anti-mouse IgG-conjugated to alkaline phosphatase was used as a secondary antibody (Bio-Rad), and the membrane was developed by a chemiluminescent detection method (New England Biolabs; Beverly, MA) (18). Densiometric quantification was performed with the use of Scion Image software.
Transmission electron microscopy and immunotransmission electron microscopy. BAEC were cultured in 100 mm tissue culture dishes and grown to confluence. Cells were exposed to laminar shear stress (19 dyn/cm2) or remained static for 1 or 3 days. After shear stress, the cells were fixed for 1 h at 4°C on ice with 1.6% paraformaldehyde and 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3), washed with 0.1 M sodium cacodylate and 3.5% sucrose buffer (pH 7.3), and then postfixed for 1 h with 1% Palade's OsO4. Cells were stained en bloc with Kellenberger's uranyl acetate, dehydrated, embedded in epoxy resin, and sectioned (28). Ultrathin sections were examined with the use of transmission electron microscopy (TEM), and random fields (each field containing part of one or two cells) were photographed. As suggested by Schnitzer et al. (35), only distinctly flask-shaped, noncoated vesicles (50–100 nm in diameter) found on the luminal and abluminal plasma membranes were scored as caveolae. Total caveolae counts were normalized to the unit length of plasma membrane measured with the use of Image Pro software (Media Cybernetics; Silver Spring, MD). We also tracked caveolae counts for the luminal or abluminal surfaces for each micrograph.
For immuno-TEM, BAEC were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 100 mM phosphate buffer (PB) (pH 7.4) for 30 min, followed by 1% paraformaldehyde in PB at 4°C overnight. The samples were rinsed four times in PB then blocked in 0.1% sodium borohydride in PB for 15 min, rinsed four times in PB, blocked a second time in 0.05% saponin, 5% BSA, 0.1% gelatin, and 5% serum in PBS for 60 min, then rinsed three times with 0.05% saponin and 0.1% BSA in PBS. The primary antibody (polyclonal caveolin-1, Transduction Laboratories) was incubated overnight at 4°C in PBS with 0.05% saponin and 0.1% BSA, then rinsed six times in PBS containing 0.05% saponin and 0.1% BSA, and incubated with the ultrasmall gold conjugated secondary antibody (Aurion; Wageningen, The Netherlands) in PBS containing 0.05% saponin and 0.1% BSA for 4 h at 4°C. Afterward, the samples were rinsed six times in PBS containing 0.05% saponin and 0.1% BSA, then three times in PBS. The samples were then postfixed with 2.0% glutaraldehyde in PB for 1 h, rinsed four times in PB, and then four times in enhancement conditioning solution (ECS) solution (Aurion). For silver enhancement, sections were agitated in a solution (R-gent SE-EM; Aurion) at room temperature for 30 min, then placed in a stop bath of 30 mM sodium thiosulfate in ECS solution for 5 min, and rinsed four times in ECS solution. The sections were then examined as described above in TEM.
Confocal immunofluorescence microscopy. After shear exposure,
cells were rinsed with PBS, then fixed and permeablized for 20 min with the
use of Cytofix/Cytoperm (PharMingen). They were rinsed twice with 1x
Perm/Wash buffer (PharMingen), quenched with 50 mM NH4Cl for 10
min, rinsed twice with PBS, and then blocked for 1 h with 3% BSA in PBS.
Circular areas (
12 mm) were isolated on the plate with the use of a
Pap Pen (Electron Microscopy Supplies; Ft. Washington, PA) and incubated with
a polyclonal antibody specific to caveolin-1 (pCav-1) or cis-Golgi
(GM130) either 1 h at RT or overnight at 4°C. Afterward, the isolated area
was rinsed with PBS, then PBS containing 3% BSA as before, followed by
treatment for 1 h at room temperature with an appropriate fluorescently
labeled secondary antibody (Alexafluor 488 for green or Alexafluor 568 for
red; Molecular Probes). The labeled cells were treated with an anti-fade
medium (Prolong, Molecular Probes) and observed with a confocal microscope
(model 510 Axiovert, Zeiss).
Chronic shear effects on subsequent acute shear activation of ERK and Akt. BAEC were exposed to 24 h of static culture or chronic shear at 10 dyn/cm2 (preconditioning) for 24 h, then the shear level was increased to 20 dyn/cm2 for a time course of 0, 5, 30, and 60 min. The static culture to 20 dyn/cm2 and 10 dyn/cm2 to 20 dyn/cm2 samples are referred to as "static-to-shear" and "shear-to-shear," respectively. Samples were analyzed with Western blots for ERK and Akt (protein kinase B) phosphorylation and total protein using antibodies specific to each protein state (Cell Signaling).
Statistical analysis. Statistical analysis was performed with the use of Student's t-test with P < 0.05 considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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These cells exposed to laminar shear or static conditions were then fixed and analyzed by TEM. As indicated by arrows in Fig. 2, plasmalemmal caveolae were found in both luminal (apical) and abluminal (basal) surfaces in cells exposed to static culture (Fig. 2, A–C) or chronic shear (Fig. 2, D–F) conditions. To determine whether shear stress changed the caveolae density, we counted the total caveolae per unit length. When BAEC were exposed to laminar shear for 1 or 3 day(s), the total number of caveolae per micron of plasma membrane significantly increased by 45% and 48%, respectively, over that of static control (1 day: static = 0.124 ± 0.013 caveolae/µm, n = 60 different micrographs taken from three different experiments; shear = 0.180 ± 0.017 caveolae/µm, n = 43, P = 0.009; 3 days: static = 0.23 ± 0.03 caveolae/µm, n = 33; shear = 0.34 ± 0.03 caveolae/µm, n = 23, P = 0.02) (Fig. 3A). The density of caveolae was higher in cells cultured for 3 days under static conditions than that of 1 day (Fig. 3A). This may be due to the time in static culture after reaching confluency. Nevertheless, shear exposure induced similar fold stimulation in caveolae density in both cases (Fig. 3A). While we quantified the total number of caveolae, we noticed a difference in their luminal and abluminal distribution. As shown in Fig. 3B, caveolae numbers per micrograph in static cultured cells were equally distributed in the luminal and abluminal surface (1 day: luminal = 2.57 ± 0.30, abluminal = 3.04 ± 0.41; 3 days: luminal = 3.42 ± 0.47, abluminal = 5.48 ± 0.97). In contrast, the caveolae number in cells exposed to chronic laminar shear increased only at the luminal surface by 80 (1 day) and 300% (3 days) over static controls (P < 0.001) with no changes in abluminal numbers (1 day: luminal = 4.64 ± 0.54, abluminal = 3.03 ± 0.32, P = 0.01; 3 days: luminal = 10.35 ± 1.01, abluminal = 3.61 ± 0.77, P < 0.001).
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To determine whether the preferred increase in luminal caveolae coincided with an increase in caveolin-1 protein, we conducted immuno-TEM with a caveolin-1 antibody. BAEC were subject to either static culture (Fig. 4A) or 24 h of laminar shear stress (Fig. 4B), then incubated with a polyclonal caveolin-1 antibody, followed by a gold particle-conjugated secondary antibody, which was silver enhanced, as shown in Fig. 4. In static cultured cells, the subcellular distribution of caveolin-1 was approximately the same for luminal and abluminal surfaces. However, when the cells were exposed to chronic laminar shear, caveolin-1 staining increased at the luminal surface. Figure 4C shows a magnified view of caveolin-1-positive vesicles, as well as single and racemose caveolae. In static cultured cells, we also observed caveolin-1 staining intracellularly (Fig. 4, A and B, arrows) clearly distinguishable from those associated with the cell surface (presumably caveolae). Because of the permeablization conditions used (0.05% saponin), it was difficult to discern the exact subcellular structure where caveolin-1 was found. These results suggest that chronic exposure of endothelial cells to laminar shear stress increases the number of caveolae and caveolin-1 preferentially on the luminal surface.
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Shear stress decreases expression of caveolin-1 mRNA and protein. Because caveolin-1 is the principle protein of caveolae, we initially hypothesized that the increase in the luminal caveolae numbers were due to increased caveolin-1 expression. To examine this hypothesis, caveolin-1 mRNA and protein expression were determined.
First, BAEC were exposed to laminar shear over a time course of up to 18 h, and caveolin-1 mRNA levels were analyzed by Northern blot analysis. To our surprise, shear exposure decreased caveolin-1 mRNA level in a time-dependent manner (Fig. 5). The decrease was obvious as early as 2 h after shear onset with a maximum 80% reduction compared with static control by 18 h (21 ± 5%, n = 3, P < 0.001) (Fig. 5, top). To examine whether the endothelial cells responded to shear in an expected manner, we also determined the mRNA levels of eNOS, a gene known to be increased by shear (8). As expected, laminar shear increased the eNOS mRNA level in a time-dependent manner, providing a positive internal control (Fig. 5, middle). The ethidium bromide staining demonstrates equal RNA loading (Fig. 5, bottom).
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Next, we examined the caveolin-1 protein level in BAEC exposed to chronic laminar shear (Fig. 6). The caveolin-1 protein level slowly decreased by 40% of static control over 3 days of continuous shear exposure (61.3 ± 6.5%, P = 0.0002) (Fig. 6, top). However, within 1 day of shear onset, no detectable difference was observed in caveolin-1 protein (Fig. 6, top left). We again observed increased eNOS protein expression in response to laminar shear, which served as a positive control (23) (Fig. 6, middle). In addition, actin protein level, which did not change in response to shear stress, was used as an additional internal control (Fig. 6, bottom).
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These results showed that chronic exposure of endothelial cells to shear stress did not increase caveolin-1 protein levels as determined by Western blot analysis. However, because immuno-TEM results (Fig. 4) indicated that shear exposure preferentially increased caveolin-1 level at the luminal surface, this led us to propose an alternative hypothesis that there is a pool of excess caveolin-1 proteins that is not used for caveolae formation in static conditions. Yet, in response to chronic shear exposure, this pool of caveolin-1 proteins may be made available to form caveolae in the luminal plasma membrane without requiring new protein synthesis.
Caveolin-1 preferentially localizes to the Golgi and plasma membrane in static and sheared BAEC, respectively. To determine whether there is an excess intracellular caveolin-1 protein pool, BAEC that were subjected to chronic shear or static control were examined by confocal microscopy with the use of a caveolin-1 antibody (Fig. 7). In static control cells, caveolin-1 was found both in the plasma membrane and perinuclear region (Fig. 7, arrows, top left). However, the majority of caveolin-1 protein was found in the perinuclear region. To determine whether the perinuclear regions corresponded to the Golgi, the cells were stained with a cis-Golgi-specific antibody (GM-130). As shown in Fig. 7, middle, the Golgi was identified as a perinuclear structure. When the two images of caveolin-1 and Golgi were merged, the perinuclear caveolin-1 staining overlapped with that of the cis-Golgi (Fig. 7, top right), suggesting their colocalization. In contrast, when BAEC were exposed to 1 day of laminar shear, caveolin-1 staining pattern changed dramatically. Instead of the pronounced perinuclear staining, caveolin-1 was mainly detected at the cell periphery, a characteristic of plasmalemmal caveolae (arrows, Fig. 7, bottom left). Merged images of caveolin-1 and the Golgi (Fig. 7, arrow, bottom middle) in shear-exposed cells displayed that caveolin-1 no longer colocalized with the Golgi marker (Fig. 7, bottom right). These results suggest that shear stress changes preferential location of caveolin-1 from the Golgi to plasma membrane. These results are consistent with the immuno-TEM data shown in Fig. 4.
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Chronic shear alters ERK and Akt activation response to subsequent acute increases in shear. Previously we (2, 13, 18, 27) have shown that a sudden exposure of static cultured cells to laminar shear stress ("static-to-shear") activates ERK and Akt (protein kinase B) in a time-dependent manner. Furthermore, we (27, 28) have shown evidence that caveolae play a critical role in shear-dependent activation of ERK. Because our current results showed that chronic exposure of endothelial cells to laminar shear stress (preconditioning) increased luminal caveolin-1 protein level and caveolae numbers, we decided to determine whether the preconditioned cells would respond to a subsequent acute change in shear stress ("shear-to-shear") differently than that of static cultured cells with the use of ERK and Akt as markers.
For this study, BAEC were preconditioned by being exposed to unidirectional laminar shear stress of 10 dyn/cm2 or remained static for 24 h. Shear stress level was then increased to 20 dyn/cm2 (acute shear) for a time course of 0, 5, 30, and 60 min (Fig. 8A). As shown in Fig. 8B, static cultured BAEC responded as expected showing a transient stimulation of ERK phosphorylation with a maximum activation by 5 min and returning to basal levels by 60 min in response to acute shear exposure (acute shear). In contrast, ERK activation of the preconditioned cells in response to the acute shear stress challenge was virtually blunted (Fig. 8B). As we have shown previously (2, 13), exposure of static cultured BAEC to acute shear increase (static-to-shear) stimulated phosphorylation of Akt in a progressive manner as a function of time reaching a maximum fourfold increase by 60 min (Fig. 8B). On the other hand, the acute shear exposure of preconditioned BAEC revealed that maximum phosphorylation of Akt occurred as early as 5 min and was maintained for as long as 60 min (Fig. 8B). To test whether the differential activation was due to differences in the changes in shear magnitude, we performed similar experiments with the exception of raising the acute shear level to 10 dyn/cm2 by using the static cultured cells. No significant difference between "0-to-10 dyn/cm2" and "0-to-20 dyn/cm2" was observed in the phosphorylation patterns of Akt and ERK (data not shown).
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These results demonstrate that acute changes in laminar shear stress level in chronically preconditioned endothelial cells blunt ERK response while accelerating the response time of another mechanosensitive signaling protein Akt.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Our TEM results showed that after 1 and 3 day(s) of exposure of endothelial cells to laminar shear, the total number of caveolae increased compared with static control. We also found that this increase was preferentially observed at the luminal surface with no significant change observed at the abluminal surface. In microvascular endothelial cells, preferential localization of caveolae to the luminal or abluminal surface has been reported in different tissues (3, 19, 39). Schnitzer et al. (34) reported that cultured microvascular endothelial cells have fewer plasmalemmal vesicles compared with endothelial cells in situ though they did not quantify the reduction. They also showed differences in the number of plasmalemmal vesicles, presumably caveolae, between cell types (bovine pulmonary aorta, vein, and microvascular endothelial cells) during culture, but it was unknown what role ambient culture conditions played (i.e., lack of shear stress) (36).
The reduction in caveolae numbers in static culture could be due to the lack of fluid flow stimulation. If this were the case, chronic shear exposure of endothelial cells could produce a more physiologically relevant environment for them. Consistent with this concept, we found that chronic exposure of BAEC to laminar shear increases the total number of caveolae. Moreover, we found that this increase in caveolae number occurred predominantly in the luminal surface, whereas it remained the same at the abluminal surface (Fig. 3). These results were consistent with our immuno-TEM data with caveolin-1 (Fig. 4).
Because the number of caveolae increased with chronic laminar shear, we expected caveolin-1 protein levels to also increase. To our surprise, we found a rapid reduction in caveolin-1 mRNA expression within 18 h (Fig. 5) and a slow and modest decrease in caveolin-1 protein levels over 3 days of shear exposure (Fig. 6). However, the reduction in caveolin-1 mRNA level in response to chronic shear is consistent with the result obtained from a DNA microarray study (21). Whereas our caveolin-1 protein result is similar to that reported by Isshiki et al. (16), our caveolin-1 mRNA results do not agree with those reported by Sun et al. (41) and Isshiki et al. (16) who used subconfluent endothelial cells. One reason that may be responsible for the discrepancy among these studies is cell confluence because it has been shown that caveolin-1 expression dramatically changes as cells reach confluency (11). Another factor could be the caveolin-1 antibodies used (polyclonal Cav-1 in our study vs. the N-20 antibody used in Sun et al.'s study). Pelkmans et al. (29) has shown previously that the N-20 antibody does not detect caveolin-1 present in the Golgi of CV-1 cells. It was also shown by Luetterforst et al. (20), that the specific antibodies raised against the NH2 terminus and caveolin scaffolding domain detected either the plasma membrane or Golgi, but not both. We have shown that the polyclonal caveolin-1 antibody binds to epitopes in both the scaffolding domain and NH2 terminus (27). This may explain our ability to detect caveolin-1 in the plasma membrane as well as in the Golgi, whereas the N-20 antibody may have excluded identification of the large Golgi pool of caveolin-1 in their immunofluorescence study, resulting in the discrepancy between the two studies.
The increased caveolae numbers without the concomitant increase in caveolin-1 protein led us to examine caveolin-1 localization using immunofluorescence and confocal microscopy. Consistent with several previous reports in static cultured endothelial cells, a major pool of caveolin-1 was found in the Golgi (12, 27, 33), and it was also found in the plasma membrane (9) (Fig. 7). Although caveolin-1 did not colocalize to the Golgi in Chinese hamster ovary cells (22), it did in other cell types, including baby hamster kidney cells, primary human fibroblasts, C2C12 (20), HeLa (42), Madin-Darby canine kidney cells (9), and CV-1 (29) cells. Luetterforst et al. (20), showed that a truncated caveolin-1 and caveolin-3 COOH terminus targets to the Golgi. They also demonstrated that this COOH terminus fused to a heterologous protein was sufficient for colocalization with the Golgi.
The preferential location of caveolin-1 in the Golgi in static cultured BAEC may be due to the lack of physical environment including shear stress existing in vivo (24). Consistent with this notion, in arterial endothelial cells in vivo, caveolin-1 is preferentially associated with the plasma membrane (36). Our study showed that chronic exposure of BAEC indeed changed the preferential localization of caveolin-1 from the Golgi to the plasma membrane caveolae. The concept of caveolin-1 translocation between plasmalemmal caveolae and the Golgi has been previously proposed in fibroblasts (40). Conrad et al. (6) showed that caveolin-1 cycles constitutively between the Golgi and the plasma membrane caveolae in both the microtubule-dependent and -independent mechanisms. Caveolin-1 proteins accumulating in the Golgi in cultured endothelial cells may represent an excess pool of caveolin-1 that is not used for caveolae formation. For unknown reasons, caveolin-1 proteins processed in the Golgi may not be transported to the plasma membrane under static culture conditions. On challenge with laminar shear force, however, the excess caveolin-1 protein may move from the Golgi to the plasma membrane forming caveolae without requiring new protein synthesis. Although chronic shear exposure decreases the caveolin-1 mRNA level to 20% of static control cells, this may be sufficient for protein production even with the increased formation of caveolae.
There are several proposed systems available to the cell for mechanotransduction. Proposed mechanosensing systems include cytoskeleton/integrins (4, 43), G proteins (14), K+ channels (26), adherens junction proteins (37), and caveolae (27, 28, 30). The preferential formation of caveolae at the luminal surface in response to chronic shear exposure strongly indicate the potential role for luminal caveolae as a major part the of mechanosensing system. Caveolae may directly sense changes in shear stress at the luminal surface. On the other hand, it is interesting to speculate that the cytoskeleton/integrins/focal adhesions may play a role in mechanosensing in the abluminal surface (15, 38, 43), whereas intercellular adherens junctions may act as a basolateral mechanosensor (37). The regionalized distribution of the mechanosensors may provide a framework for the decentralization hypothesis that was proposed by Davies and colleague (7, 15), stating that for a given stress configuration, several intracellular mechanisms respond and integrate a cellular response.
The luminal formation of caveolae and caveolin-1 may have a major impact on how the cells respond to the subsequent mechanical or humoral challenges. For example, endothelial cells grown under chronic shear conditions may respond differently to a subsequent physical or humoral stimulus compared with that of static cultured cells. Indeed, unlike with static cultured cells, we found that endothelial cells exposed to chronic laminar shear conditions (preconditioning) have a blunted ERK phosphorylation, whereas Akt activation is accelerated in response to the subsequent increase in shear stress (acute shear).
It is well known that laminar shear is atheroprotective and inhibits endothelial proliferation, whereas low and unstable shear are proatherogenic (7). It has been somewhat curious why exposure of static cultured cells to laminar shear stimulates ERK activity, which is normally linked to proliferative responses (18). Most endothelial cells that are exposed to laminar shear in straight part of arteries have very low cell turnover rate, and those that do are usually found in atherosclerotic lesion-prone areas (7). Endothelial cells in lesion-prone areas around branched or curved arteries are exposed to low and disturbed shear conditions (7). Therefore, it is tempting to speculate that the ERK response to acute shear in static cells (static to shear) may represent a response of those in lesion-prone areas. In contrast, endothelial cells preconditioned to laminar shear may represent those found in straight arteries showing very low cell proliferation. It is also interesting to note that endothelium in lesion-prone areas show a significantly higher level of apoptosis than those in straight arteries (31). Because Akt is a key antiapoptotic molecule, our result showing the accelerated response in shear preconditioned cells is consistent with the antiapoptotic effect of laminar shear.
At present, it is not known why chronic shear exposure differentially altered the cell-signaling responses of ERK and Akt in response to a subsequent shear level change (acute shear). This may be due to changes in formation or interaction of cell-signaling complexes in and around caveolae-like domains. There may be an optimum range of caveolin-1 level that is required for proper regulation of mechanosensitive activation of ERK. Chronic shear exposure may regulate caveolae number and location for an optimal activation of cell signaling pathways. Previously, we have shown that removal of plasma membrane cholesterol using cyclodextrin or filipin, or inhibition of caveolin-1 by a neutralizing antibody blocks shear stress-dependent activation of ERK in static cultured cells (27, 28), demonstrating a critical role of caveolin-1 and caveolae in this mechanoresponse. As discussed above, in static cultured cells, there seems to be an excess pool of caveolin-1 in intracellular structures such as the Golgi, whereas a relatively small amount of caveolin-1 is in caveolae. This distribution may have allowed for a transient and robust activation of ERK in a caveolae-dependent manner. On the other hand, chronic shear exposure induces relocation of caveolin-1 and caveolae formation to the luminal cell surface. Because caveolin-1 is a negative regulator of the ERK pathway (10), relative increase in caveolin-1 and caveolae induced by chronic shear may have resulted in blocking ERK activation by an acute shear. Alternatively, chronic shear exposure may have induced a desensitization mechanism that blocks further activation of ERK in response to the acute shear.
In summary, we have shown here that chronic shear exposure controls the formation and localization of caveolae and caveolin-1 in endothelial cells. We suggest that the luminal caveolae formation plays a critical role in mechanosensing and subsequent mechanosensitive cellular responses.
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DISCLOSURES |
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Present address for H. Park: Dan Kook University, Dept. of Molecular Biology, Room 506, Kwa-Hak Kwan, San 8, Hannamdong, Yongsan-Gu, Seoul 140-714, Korea.
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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/
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J Biol Chem 272:
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 S. J. Nixon, A. Carter, J. Wegner, C. Ferguson, M. Floetenmeyer, J. Riches, B. Key, M. Westerfield, and R. G. Parton Caveolin-1 is required for lateral line neuromast and notochord development J. Cell Sci., July 1, 2007; 120(13): 2151 - 2161. [Abstract] [Full Text] [PDF]
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 M. O. Platt, R. F. Ankeny, G.-P. Shi, D. Weiss, J. D. Vega, W. R. Taylor, and H. Jo Expression of cathepsin K is regulated by shear stress in cultured endothelial cells and is increased in endothelium in human atherosclerosis Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1479 - H1486. [Abstract] [Full Text] [PDF]
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F. Shi, Y.-J. Chiu, Y. Cho, T. A. Bullard, M. Sokabe, and K. Fujiwara Down-regulation of ERK but not MEK phosphorylation in cultured endothelial cells by repeated changes in cyclic stretch Cardiovasc Res, March 1, 2007; 73(4): 813 - 822. [Abstract] [Full Text] [PDF]
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 A. Zulli, B. F. Buxton, M. J. Black, Z. Ming, A. Cameron, and D. L. Hare The Immunoquantification of Caveolin-1 and eNOS in Human and Rabbit Diseased Blood Vessels J. Histochem. Cytochem., February 1, 2006; 54(2): 151 - 159. [Abstract] [Full Text] [PDF]
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 A. C. Bellott, K. C. Patel, and T. J. Burkholder Reduction of caveolin-3 expression does not inhibit stretch-induced phosphorylation of ERK2 in skeletal muscle myotubes J Appl Physiol, April 1, 2005; 98(4): 1554 - 1561. [Abstract] [Full Text] [PDF]
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 P. M. Bauer, J. Yu, Y. Chen, R. Hickey, P. N. Bernatchez, R. Looft-Wilson, Y. Huang, F. Giordano, R. V. Stan, and W. C. Sessa Endothelial-specific expression of caveolin-1 impairs microvascular permeability and angiogenesis PNAS, January 4, 2005; 102(1): 204 - 209. [Abstract] [Full Text] [PDF]
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 J.-P. Gratton, P. Bernatchez, and W. C. Sessa Caveolae and Caveolins in the Cardiovascular System Circ. Res., June 11, 2004; 94(11): 1408 - 1417. [Abstract] [Full Text] [PDF]
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 J. T. Ferraro, M. Daneshmand, R. Bizios, and V. Rizzo Depletion of plasma membrane cholesterol dampens hydrostatic pressure and shear stress-induced mechanotransduction pathways in osteoblast cultures Am J Physiol Cell Physiol, April 1, 2004; 286(4): C831 - C839. [Abstract] [Full Text] [PDF]
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