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Am J Physiol Heart Circ Physiol 285: H1113-H1122, 2003. First published May 22, 2003; doi:10.1152/ajpheart.00302.2003
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Chronic shear induces caveolae formation and alters ERK and Akt responses in endothelial cells

Nolan L. Boyd,1 Heonyong Park,3 Hong Yi,2 Yong Chool Boo,1 George P. Sorescu,1 Michelle Sykes,1 and Hanjoong Jo1,3

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


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Caveolae are plasmalemmal domains enriched with cholesterol, caveolins, and signaling molecules. Endothelial cells in vivo are continuously exposed to shear conditions, and their caveolae density and location may be different from that of static cultured cells. Here, we show that chronic shear exposure regulates formation and localization of caveolae and caveolin-1 in bovine aortic endothelial cells (BAEC). Chronic exposure (1 or 3 days) of BAEC to laminar shear increased the total number of caveolae by 45–48% above static control. This increase was due to a rise in the luminal caveolae density without changing abluminal caveolae numbers or increasing caveolin-1 mRNA and protein levels. Whereas some caveolin-1 was found in the plasma membrane in static-cultured cells, it was predominantly localized in the Golgi. In contrast, chronic shear-exposed cells showed intense caveolin-1 staining in the luminal plasma membrane with minimum Golgi association. The preferential luminal localization of caveolae may play an important role in endothelial mechanosensing. Indeed, we found that chronic shear exposure (preconditioning) altered activation patterns of two well-known shear-sensitive signaling molecules (ERK and Akt) in response to a step increase in shear stress. ERK activation was blunted in shear preconditioned cells, whereas the Akt response was accelerated. These results suggest that chronic shear stimulates caveolae formation by translocating caveolin-1 from the Golgi to the luminal plasma membrane and alters cell signaling responses.

caveolin-1; mechanosensing; atherosclerosis

SHEAR STRESS REGULATES vascular endothelial cells both acutely and chronically by controlling production of autocrine and paracrine vasoactive factors (7). Through these endothelial responses, shear stress controls vascular tone, vessel wall remodeling, binding of blood circulating cells to endothelium, and hemostasis (7). Shear stress selectively and differentially regulates expression of many genes that are important in pathophysiology of vessel wall function (5). Induction of specific genes by shear stress in endothelial cells is mediated by the activation of mechanosensitive signaling pathways that include at least three members of the mitogen-activated protein (MAP) kinase family: extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), and big MAP kinase 1 (18, 44).

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Cell culture. BAEC harvested from descending thoracic aortas were maintained (37°C, 5% CO2) in a growth medium [DMEM (1 g/l glucose, Mediatech) containing 20% FCS (Atlanta Biologicals) without antibiotics] (17). Cells used in this study were between passages 3 and 10.

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 [{alpha}-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 (~{varphi}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.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Chronic laminar shear exposure aligns endothelial cell shape and changes density and subcellular location of caveolae. To determine whether chronic exposure of endothelial cells under shear stress conditions regulates the formation and subcellular location of caveolae, BAEC monolayers were exposed to laminar shear or static conditions for 1 or 3 day(s), and examined by light microscopy and TEM. As expected, the BAECs grown in static conditions showed typical polygonal cobblestone shapes, whereas the cells subjected to laminar shear (19 dyn/cm2) for 1 day showed alignment toward the imposed flow direction (Fig. 1). The same effect on alignment was observed by 3 days of laminar shear exposure (data not shown).



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  		Fig. 1. Laminar shear stress aligns bovine aortic endothelial cells (BAEC). Confluent BAEC monolayers grown in 10-cm dishes under static conditions were exposed to static (A) or unidirectional laminar shear (19 dyn/cm2) conditions (B) or for 1 day. Cells were then observed by phase microscopy. Representative micrographs (original magnification, x10) of endothelial cell monolayers are shown. Note the typical cobblestone appearance for static cultured cells showing no directional alignment (A), whereas cells become aligned to the direction of shear stress (B). Arrow shows alignment of flow direction.

 

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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  		Fig. 2. Laminar shear stress increases luminal caveolae density. Confluent BAEC monolayers exposed to static conditions or laminar shear stress for 1 or 3 day(s) as in Fig. 1, were fixed and examined by transmission electron microscopy (TEM). Representative electron micrographs of BAEC exposed to static conditions (A) and shear stress (B) are shown. A and D show both luminal and abluminal surfaces of cells (bar = 2 µm). Shown in B, C, E, and F are magnified views of A and D clearly revealing caveolae on luminal (B and E) and abluminal (C and F) surfaces (arrows). The numbers and location of caveolae and the cell surface length (in µm) were determined from the micrographs with the use of Image Pro software. N, nucleus; L, lumen; BM, basement membrane.

 


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  		Fig. 3. Laminar shear increases caveolae at the luminal surface. A: total number of cellular caveolae were counted for each TEM micrograph and normalized to the cell's perimeter length (in µm) for both static culture (1 day: n = 60, 3 days: n = 34, n = the number of micrographs taken from three different cell experiments) and laminar shear (1 day: n = 43, 3 days: n = 23), {dagger}P = 0.009, {dagger}{dagger}P = 0.02. B: for each micrograph, caveolae were categorized by localization to the luminal or abluminal surface for both static culture and 24 h of laminar shear; *P < 0.001, **P = 0.01. L, luminal surface; Abl, abluminal surface. Values are means ± SE.

 

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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  		Fig. 4. Immuno-TEM demonstrates shear-dependent luminal localization of caveolin-1. BAEC were subject to static culture (A) or shear (B) for 24 h then incubated with a caveolin-1 polyclonal antibody. Ultrasmall gold particles were conjugated to the secondary antibody, then silver enhanced for better identification. C is a magnified view of box in B showing caveolin-1 staining associated with caveolae (arrowheads) and internal vesicles (#). Shown micrographs are representative of 30–45 cells obtained from two independent experiments. Bar = 800 nm (A and B), and 200 nm (C).

 

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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  		Fig. 5. Shear stress decreases expression of caveolin-1 mRNA in a time-dependent manner. BAEC were subject to a shear stress (S) over a time course of up to 18 h, then examined for caveolin-1 (Cav-1) mRNA by Northern blot analysis (top). Endothelial nitric oxide synthase (eNOS) mRNA (middle), and the ethidium bromide (Et.Br.) blot (bottom) were used as a positive and equal RNA loading for each well controls, respectively. The graph represents the accumulated data for the time course of the shear effects on caveolin-1 mRNA as a percentage of control (t = 0) and normalized to 18S rRNA. Values are means ± SE (n = 3 to 5; *P < 0.05).

 

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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  		Fig. 6. Shear stress decreases caveolin-1 protein expression. BAEC were exposed to shear or static control for 1 and 3 days and 20 µg of total cell lysate used for Western protein analysis per sample. Total caveolin-1 was immunoblotted with the monoclonal antibody 2297, which detects both the {alpha}- and {beta}-isoforms (top). To verify the caveolin-1 Western blot results, the same blot was probed for eNOS (middle) and actin (bottom) as positive and equal loading controls, respectively. The bar graph represents the accumulated data of at least three independent experiments as a percentage of control. Values are means ± SE (n = 3 to 5, *P < 0.001).

 

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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  		Fig. 7. Caveolin-1 preferentially localizes to the Golgi and plasma membrane in static and sheared BAEC, respectively. Dual-labeled confocal microscopy was used to detect the localization of caveolin-1 with the use of antibodies specific for caveolin-1 (p-Cav-1) and cis-Golgi (GM-130) in static cultured cells and cells exposed to 24 h of laminar shear stress. A: in static culture cells (top), caveolin-1 (left) staining pattern shows a punctate, peripheral pattern representative of the plasma membrane and a perinuclear pool (arrows). The cis-Golgi marker (arrows) is shown in the same cells (middle). Right, merged images showing the colocalization of the perinuclear caveolin-1 pool and the cis-Golgi (right). However, after 24 h of shear (bottom), the perinuclear pool is undetectable, whereas caveolin-1 (arrows) preferentially localizes in the plasma membrane (left). The cis-Golgi is still detected in a perinuclear pattern (middle) that when merged with caveolin-1 is no longer colocalized (right). The images are representative of at least 5 independent experiments (bar = 10 µm).

 

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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  		Fig. 8. Chronic shear exposure alters activation patterns of ERK and Akt by a subsequent step shear change (acute shear). BAEC were exposed to laminar shear (10 dyn/cm2) or static control for 1 day. The static cultured cells were then exposed to an acute shear ("0-to-20" dyn/cm2) for a time course of 5, 30, and 60 min, whereas the chronically sheared (preconditioned) cells were exposed to an acute shear ("10-to-20" dyn/cm2) for the same time course (A). Activation of ERK and Akt was analyzed by Western blot with the use of cell lysates with antibodies to phosphorylated forms of ERK and Akt, respectively, and quantitated by densitometry. Line graphs represent means ± SE (n = 3–5; * and **P < 0.05). Membranes probed with phosphorylated ERK and Akt were stripped and reprobed with antibodies specific to total ERK and Akt, respectively, as internal controls.

 

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.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The main and novel findings of this report are that 1) laminar shear exposure increases the number of caveolae preferentially at the luminal surface, 2) the increase in luminal caveolae density are not mediated by increasing overall caveolin-1 gene and protein expression levels, 3) laminar shear changes preferential location of caveolin-1 from the Golgi to the plasma membrane caveolae, and 4) chronic shear alters ERK and Akt activation response to subsequent acute increases in shear.

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.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-71014 and HL-67413 and National Aeronautics and Space Administration Grant NAG2-1348 (to H. Jo).

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.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Jo, Wallace H. Coulter Dept. of Biomedical Engineering at Georgia Tech and Emory Univ., 308D WMB, Atlanta, GA 30322 (E-mail: hanjoong.jo{at}bme.gatech.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Anderson RG. The caveolae membrane system. Annu Rev Biochem 67: 199–225, 1998.[ISI][Medline]
  2. Boo YC, Sorescu G, Boyd N, Shiojima I, Walsh K, Du J, and Jo H. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A. J Biol Chem 277: 3388–3396, 2002.[Abstract/Free Full Text]
  3. Bruns RR and Palade GE. Studies on blood capillaries. I. General organization of blood capillaries in muscle. J Cell Biol 37: 244–276, 1968.[Abstract/Free Full Text]
  4. Chen KD, Li YS, Kim M, Li S, Yuan S, Chien S, and Shyy JY. Mechanotransduction in response to shear stress. Roles of receptor tyrosine kinases, integrins, and Shc. J Biol Chem 274: 18393–18400, 1999.[Abstract/Free Full Text]
  5. Chien S, Li S, and Shyy YJ. Effects of mechanical forces on signal transduction and gene expression in endothelial cells. Hypertension 31: 162–169, 1998.[Abstract/Free Full Text]
  6. Conrad PA, Smart EJ, Ying YS, Anderson RG, and Bloom GS. Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule-dependent and microtubule-independent steps. J Cell Biol 131: 1421–1433, 1995.[Abstract/Free Full Text]
  7. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev 75: 519–560, 1995.[Abstract/Free Full Text]
  8. Davis ME, Cai H, Drummond GR, and Harrison DG. Shear stress regulates endothelial nitric oxide synthase expression through c-Src by divergent signaling pathways. Circ Res 89: 1073–1080, 2001.[Abstract/Free Full Text]
  9. Dupree P, Parton RG, Raposo G, Kurzchalia TV, and Simons K. Caveolae and sorting in the trans-Golgi network of epithelial cells. EMBO J 12: 1597–1605, 1993.[ISI][Medline]
  10. Engelman JA, Chu C, Lin A, Jo H, Ikezu T, Okamoto T, Kohtz DS, and Lisanti MP. Caveolin-mediated regulation of signaling along the p42/44 MAP kinase cascade in vivo. A role for the caveolin-scaffolding domain. FEBS Lett 428: 205–211, 1998.[ISI][Medline]
  11. Galbiati F, Volonte D, Engelman JA, Watanabe G, Burk R, Pestell RG, and Lisanti MP. Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade. EMBO J 17: 6633–6648, 1998.[ISI][Medline]
  12. Garcia-Cardena G, Martasek P, Masters BS, Skidd PM, Couet J, Li S, Lisanti MP, and Sessa WC. Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the NOS caveolin binding domain in vivo. J Biol Chem 272: 25437–25440, 1997.[Abstract/Free Full Text]
  13. Go YM, Boo YC, Park H, Maland MC, Patel R, Pritchard KA Jr, Fujio Y, Walsh K, Darley-Usmar V, and Jo H. Protein kinase B/Akt activates c-Jun NH2-terminal kinase by increasing NO production in response to shear stress. J Appl Physiol 91: 1574–1581, 2001.[Abstract/Free Full Text]
  14. Gudi SR, Clark CB, and Frangos JA. Fluid flow rapidly activates G proteins in human endothelial cells. Involvement of G proteins in mechanochemical signal transduction. Circ Res 79: 834–839, 1996.[Abstract/Free Full Text]
  15. Helmke BP and Davies PF. The cytoskeleton under external fluid mechanical forces: hemodynamic forces acting on the endothelium. Ann Biomed Eng 30: 284–296, 2002.[ISI][Medline]
  16. Isshiki M, Ando J, Yamamoto K, Fujita T, Ying Y, and Anderson RG. Sites of Ca2+ wave initiation move with caveolae to the trailing edge of migrating cells. J Cell Sci 115: 475–484, 2002.[Abstract/Free Full Text]
  17. Jo H, Dull RO, Hollis TM, and Tarbell JM. Endothelial albumin permeability is shear dependent, time dependent, and reversible. Am J Physiol Heart Circ Physiol 260: H1992–H1996, 1991.[Abstract/Free Full Text]
  18. Jo H, Sipos K, Go YM, Law R, Rong J, and McDonald JM. Differential effect of shear stress on extracellular signal-regulated kinase and N-terminal Jun kinase in endothelial cells. Gi2- and G{beta}/{gamma}-dependent signaling pathways. J Biol Chem 272: 1395–1401, 1997.[Abstract/Free Full Text]
  19. Johansson BR. Quantitative ultrastructural morphometry of blood capillary endothelium in skeletal muscle. Effect of venous pressure. Microvasc Res 17: 118–130, 1979.[ISI][Medline]
  20. Luetterforst R, Stang E, Zorzi N, Carozzi A, Way M, and Parton RG. Molecular characterization of caveolin association with the Golgi complex: identification of a cis-Golgi targeting domain in the caveolin molecule. J Cell Biol 145: 1443–1459, 1999.[Abstract/Free Full Text]
  21. McCormick SM, Eskin SG, McIntire LV, Teng CL, Lu CM, Russell CG, and Chittur KK. DNA microarray reveals changes in gene expression of shear stressed human umbilical vein endothelial cells. Proc Natl Acad Sci USA 98: 8955–8960, 2001.[Abstract/Free Full Text]
  22. Mundy DI, Machleidt T, Ying YS, Anderson RG, and Bloom GS. Dual control of caveolar membrane traffic by microtubules and the actin cytoskeleton. J Cell Sci 115: 4327–4339, 2002.[Abstract/Free Full Text]
  23. Nishida K, Harrison DG, Navas JP, Fisher AA, Dockery SP, Uematsu M, Nerem RM, Alexander RW, and Murphy TJ. Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase. J Clin Invest 90: 2092–2096, 1992.[ISI][Medline]
  24. Oh P and Schnitzer JE. Isolation and subfractionation of plasma membranes to purify caveolae separately from glycosyl-phosphatidylinositol-anchored protein microdomains. In: Cell Biology: A Laboratory Handbook, edited by Celis JE. San Diego, CA: Academic, 1998, p. 34–45.
  25. Okamoto T, Schlegel A, Scherer PE, and Lisanti MP. Caveolins, a family of scaffolding proteins for organizing "preassembled signaling complexes" at the plasma membrane. J Biol Chem 273: 5419–5422, 1998.[Free Full Text]
  26. Olesen SP, Clapham DE, and Davies PF. Haemodynamic shear stress activates a K+ current in vascular endothelial cells. Nature 331: 168–170, 1988.[Medline]
  27. Park H, Go YM, Darji R, Choi JW, Lisanti MP, Maland MC, and Jo H. Caveolin-1 regulates shear stress-dependent activation of extracellular signal-regulated kinase. Am J Physiol Heart Circ Physiol 278: H1285–H1293, 2000.[Abstract/Free Full Text]
  28. Park H, Go YM, St. John PL, Maland MC, Lisanti MP, Abrahamson DR, and Jo H. Plasma membrane cholesterol is a key molecule in shear stress-dependent activation of extracellular signal-regulated kinase. J Biol Chem 273: 32304–32311, 1998.[Abstract/Free Full Text]
  29. Pelkmans L, Kartenbeck J, and Helenius A. Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nat Cell Biol 3: 473–483, 2001.[ISI][Medline]
  30. Rizzo V, Sung A, Oh P, and Schnitzer JE. Rapid mechanotransduction in situ at the luminal cell surface of vascular endothelium and its caveolae. J Biol Chem 273: 26323–26329, 1998.[Abstract/Free Full Text]
  31. Rossig L, Dimmeler S, and Zeiher AM. Apoptosis in the vascular wall and atherosclerosis. Basic Res Cardiol 96: 11–22, 2001.[ISI][Medline]
  32. Sambrook J, Fritsch EF, and Maniatis T. Extraction, purification, and analysis of messenger RNA from eukaryotic cells. In: Molecular Cloning, A Laboratory Manual, edited by Nolan C. Plainview, NY: Cold Spring Harbor Laboratory, 1989, p. 43–45.
  33. Schlegel A and Lisanti MP. A molecular dissection of caveolin-1 membrane attachment and oligomerization. Two separate regions of the caveolin-1 C-terminal domain mediate membrane binding and oligomer/oligomer interactions in vivo. J Biol Chem 275: 21605–21617, 2000.[Abstract/Free Full Text]
  34. Schnitzer JE, Carley WW, and Palade GE. Specific albumin binding to microvascular endothelium in culture. Am J Physiol Heart Circ Physiol 254: H425–H437, 1988.[Abstract/Free Full Text]
  35. Schnitzer JE, Oh P, Pinney E, and Allard J. Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. J Cell Biol 127: 1217–1232, 1994.[Abstract/Free Full Text]
  36. Schnitzer JE, Siflinger-Birnboim A, Del Vecchio PJ, and Malik AB. Segmental differentiation of permeability, protein glycosylation, and morphology of cultured bovine lung vascular endothelium. Biochem Biophys Res Commun 199: 11–19, 1994.[ISI][Medline]
  37. Shay-Salit A, Shushy M, Wolfovitz E, Yahav H, Breviario F, Dejana E, and Resnick N. VEGF receptor 2 and the adherens junction as a mechanical transducer in vascular endothelial cells. Proc Natl Acad Sci USA 99: 9462–9467, 2002.[Abstract/Free Full Text]
  38. Shyy JY and Chien S. Role of integrins in endothelial mechanosensing of shear stress. Circ Res 91: 769–775, 2002.[Abstract/Free Full Text]
  39. Simionescu M, Simionescu N, and Palade GE. Morphometric data on the endothelium of blood capillaries. J Cell Biol 60: 128–152, 1974.[Abstract/Free Full Text]
  40. Smart EJ, Ying YS, Conrad PA, and Anderson RG. Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation. J Cell Biol 127: 1185–1197, 1994.[Abstract/Free Full Text]
  41. Sun RJ, Muller S, Stoltz JF, and Wang X. Shear stress induces caveolin-1 translocation in cultured endothelial cells. Eur Biophys J 30: 605–611, 2002.[ISI][Medline]
  42. Thomsen P, Roepstorff K, Stahlhut M, and van Deurs B. Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking. Mol Biol Cell 13: 238–250, 2002.[Abstract/Free Full Text]
  43. Wang N, Butler JP, and Ingber DE. Mechanotransduction across the cell surface and through the cytoskeleton. Science 260: 1124–1127, 1993.[Abstract/Free Full Text]
  44. Yan C, Takahashi M, Okuda M, Lee JD, and Berk BC. Fluid shear stress stimulates big mitogen-activated protein kinase 1 (BMK1) activity in endothelial cells. Dependence on tyrosine kinases and intracellular calcium. J Biol Chem 274: 143–150, 1999.[Abstract/Free Full Text]
  45. Zhang W, Razani B, Altschuler Y, Bouzahzah B, Mostov KE, Pestell RG, and Lisanti MP. Caveolin-1 inhibits epidermal growth factor-stimulated lamellipod extension and cell migration in metastatic mammary adenocarcinoma cells (MTLn3). Transformation suppressor effects of adenovirus-mediated gene delivery of caveolin-1. J Biol Chem 275: 20717–20725, 2000.[Abstract/Free Full Text]



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