Caveolin-1 Facilitates Mechanosensitive Protein Kinase B (Akt) Signaling In Vitro and In Vivo
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循环研究杂志 2005年第4期
the Department of Internal Medicine I/Cardiology (D.G.S., J.H., H.T), Lung Center (U.S., O.E.)
Institute for Anatomy and Cell Biology (W.K.), Giessen University, Giessen, Germany
the Department of Internal Medicine II/Cardiology (C.S., R.H.S., R.C.B.-D.), Dresden University of Technology, Dresden, Germany.
Abstract
Mechanotransduction represents an integral part of vascular homeostasis and contributes to vascular lesion formation. Previously, we demonstrated a mechanosensitive activation of phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt) resulting in p27Kip1 transcriptional downregulation and cell cycle entry of vascular smooth muscle cells (VSMC). In this study, we further elucidated the signaling from outside-in toward PI3-K/Akt in vitro and in an in vivo model of elevated tensile force. When VSMC were subjected to cyclic stretch (0.5 Hz at 125% resting length), PI3-K, Akt, and Src kinases were found activated. Disrupting caveolar structures with -cyclodextrin or transfection of VSMC with caveolin-1 antisense oligonucleotides (ODN) prevented PI3-K and Akt activation and cell cycle entry. Furthermore, PI3-K and Akt were resistant to activation when Src kinases were inhibited pharmacologically or by overexpression of a kinase-dead c-Src mutant. V3 integrins were identified to colocalize with PI3-K/caveolin-1 complexes, and blockade of V3 integrins prevented Akt activation. The central role of caveolin-1 in mechanotransduction was further examined in an in vivo model of elevated tensile force. Interposition of wild-type (WT) jugular veins into WT carotid arteries resulted in a rapid Akt activation within the veins that was almost abolished when veins of caveolin-1 knockout (KO) mice were used. Furthermore, late neointima formation within the KO veins was significantly reduced. Our study provides evidence that PI3-K/Akt is critically involved in mechanotransduction of VSMC in vitro and within the vasculature in vivo. Furthermore, caveolin-1 is essential for the integrin-mediated activation of PI3-K/Akt.
Key Words: remodeling muscle, smooth signal transduction stress vasculature
Introduction
It is currently recognized that the machinery governing the cell cycle regulates multiple cellular functions in the cardiovascular system, thereby maintaining the homeostasis of the vasculature and allowing its adaptation to acute and chronic changes. Besides organizing cellular proliferation, the cell cycle is involved in migration, apoptosis, and hypertrophy.1
One of the major constituents of the blood vessel wall responsible for the maintenance of vessel structures and functions are vascular smooth muscle cells (VSMCs). In the vasculature, VSMCs are constantly exposed to alternating mechanical forces. Under normal tensile stress, VSMCs are relatively insensitive to mitogens. During altered mechanical stress (eg, high blood pressure), however, VSMCs upregulate protein synthesis in response to growth factors, dedifferentiate, and increase their proliferative rate, resulting in medial hypertrophy and intimal hyperplasia.2 Whereas the commonly accepted "response to injury" hypothesis suggests that growth factors are locally released, thereby initiating cell cycle entry and progression of vascular cells, the signaling pathways arising solely from mechanical force have just partially come to light.3 Recently, we demonstrated that the earliest cell cycle events can occur in a mechanosensitive fashion independently of newly released or synthesized growth factors but that they are dependent on an intact integrin signaling.4
Besides mediating cell adhesion, integrins transmit extracellular stimuli into intracellular signaling events.5 Thus, integrins mediate VSMC dedifferentiation, migration, proliferation, and apoptosis.6 Signaling through integrins requires physical interaction with other membrane proteins and subsequent association with signal transduction proteins of the cytoplasm. Caveolin-1 functions as a membrane adaptor. For example, caveolin-1 links the integrin -subunit to the c-Src kinase pathway and subsequently to the mitogen-activated protein kinase pathway (MAPK) to promote cell cycle progression.7 Another pathway critical for VSMC proliferation consists of the phosphoinositide 3-kinase(PI3-K)/protein kinase B (Akt) signal transduction pathway.8 Although the MAPK pathway had previously been shown to be responsive to mechanical force,9 we were recently able to demonstrate that cyclic stretch resulted in a rapid PI3-K/Akt activation as well, thereby inducing forkhead transcription factoreCdependent downregulation of the cell cycle inhibitor p27Kip1.4 This chain of events facilitated mechanosensitive cell cycle entry and proliferation of VSMCs in contrast to the posttranscriptional downregulation of p27Kip1 protein in VSMCs stimulated with serum mitogens. In this way, p27Kip1 may serve as a differential sensor for growth factoreC and mechanically induced cell cycle entry in VSMCs. In the present study, we further elucidated the integrin/adaptor protein interactions resulting in growth factoreCindependent PI3-K/Akt activation and subsequent cell cycle entry. We present evidence that the structural protein caveolin-1 is essential for integrin-mediated PI3-K/Akt activation during cyclic stretch of VSMCs in vitro and in an in vivo model of elevated tensile force. In our in vitro model, mechanical force induces the formation of functional signaling complexes composed of V3 integrins, caveolin-1, PI3-K/Akt, and the nonreceptor tyrosine kinase c-Src, resulting in PI3-K/Akt activation and thereby facilitating a mechanosentive proliferative response of VSMCs.
Materials and Methods
Cell Culture, Stretch Apparatus, and Experimental Conditions
Primary cultures of VSMCs were initiated by enzymatic dissociation from the aorta of 7- to 8-week-old male Sprague-Dawley rats (Charles River Breeding Laboratories, Kingston, NY).10 The cells were seeded (10 000 cells/cm2) onto 6-well fibronectin-coated FlexI plates (Flexcell). Studies were conducted on VSMCs (passages 7 to 12) after achieving confluence in 10% FCS/DMEM/F12, followed by serum withdrawal for 2 days to achieve quiescence. On the day of the experiment, fresh serum-free medium was substituted and uniaxial cyclic stretch was applied with a flexercell apparatus (125% resting length, 0.5 Hz) in a tissue culture incubator.
Immunocytochemistry
VSMCs were grown on fibronectin-coated flexercell wells. Quiescent or stretched (30 minutes) cells were fixed for 10 minutes in ice-cold acetone. Cells were covered for 20 minutes with 10% normal goat serum, followed by incubation with rabbit anti-caveolin-1 (1:100) and mouse anti PI3-K (p85, 1:50) for 1 hour in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin. After two washing steps (10 minutes in PBS), cells were incubated with secondary antibodies for 40 minutes: donkey anti-rabbit IgG conjugated to FITC (1:200) and goat anti-mouse IgG conjugated to Alexa Fluor 546 (1:200). After washing, cells were mounted in Vectashield mounting medium H-1000 containing DAPI (5 e/mL, Linaris) and evaluated using an epifluorescence microscope (DMRB, Leica). Negative controls were performed using only the secondary antibody.
Preparation of Cellular Lysates and Immunoblot Analysis
Specific protein content in cell lysates was analyzed by immunoblot as previously described.11 Briefly, cell lysis buffer contained 50 mmol/L HEPES pH 7.4, 100 mmol/L NaCl, 50 mmol/L NaF, 5 mmol/L glycerophosphate, 2 mmol/L EDTA, 2 mmol/L EGTA, 1 mmol/L vanadat, 0.5% octyglucopyranoside, PMSF 100 e/mL, aprotinin 30 e蘈/mL, leupeptin 1 ng/mL, and okadaic acid 10 eol/L. Lysate proteins (20 e) were run on a polyacrylamide gel and blotted onto nitrocellulose (Hybond-ECL, Amersham). After blocking, blots were incubated with primary antibody for 1 hour at room temperature. Specific proteins were detected by enhanced chemiluminescence (ECL+, Amersham) after labeling with horseradish peroxidase-labeled secondary antibody (1:2000 for 1 hour) according to the manufacturer’s instructions.
Flow Cytometry
Cells were harvested by trypsinization, fixed overnight with 75% methanol, washed, and incubated with 100 e/mL RNase (Oncogene Research Products) and 10 e/mL propidium iodide in PBS for 1 hour at 37°C. Samples were analyzed for DNA content using a high-speed cell sorter (EPICs Altra, Beckman Coulter). Data were computer-analyzed with commercially available software (Multicycle, Phoenix Flow Systems).
Mice and Vein Graft Procedure
Caveolin-1eC/eC mice were provided by M. Drab, Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany. All animal experiments were performed according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals (Giessen University). The vein graft procedure has been described previously.12 Briefly, mice were anesthetized with ketamine (50 mg/kg body weight, IP). The jugular vein was harvested from the donor. In the recipient, the left common carotid artery was mobilized from proximal to the distal bifurcation, cut in the middle, and a cuff (0.63 mm outside diameter and 0.5 mm inside diameter, Portex LTD) placed at each end. At each end, the artery was turned inside out over the cuff and ligated. The vein segment was grafted between the two ends of the carotid artery by sleeving the ends of the vein over the artery-cuff and ligating them together with an 8-0 suture.
For Western blot experiments, vein grafts were perfused with PBS, excised immediately, and snap-frozen in liquid nitrogen until further use.
Statistical Analysis
Data are given as mean±SEM. Statistical analysis was performed by ANOVA. Post hoc analysis was performed by the method of Bonferroni. All experiments, including the immunoblots, were independently repeated at least three times.
For detailed description of reagents, transfection procedures, magnetic activated cell sorting (MACS), phosphoinositide 3-kinase assay, Src family kinase assay, and histological evaluation of tissue sections please refer to the online data supplement available at http://circres.ahajournals.org.
Results
Mechanical Strain Induces Colocalization of PI3-K With Caveolin-1 and Their Clustering at Focal Adhesion Sites
Previously, we had demonstrated that stretch-induced cell cycle entry and progression can be prevented by pharmacological inhibition of PI3-K (LY294002 and wortmannin) or overexpression of a constitutive negative Akt, indicating a requirement for PI3-K/Akt in mechanically induced proliferation of VSMCs.4 In the present examination of cellular distribution, it was found that in quiescent cells, PI3-K had a homogeneous cytosolic distribution and that caveolin-1 was distributed at the cell surface in a diffuse, punctuate manner (Figure 1A and 1B). After 15 minutes of cyclic stretch, PI3-K and caveolin-1 accumulated in clusters at the cell membrane. Double-staining suggested their colocalization (Figure 1A and 1B). The PI3-K/caveolin-1 complexes seemed to be located at focal adhesion sites as implicated by their colocalization with vinculin (shown for caveolin-1; Figure 1A and 1B).
Mechanosensitive Activation of PI3-K/Akt Requires Intact Caveolar Structures and Caveolin-1
We examined whether caveolar structures are required for the mechanosensitive activation of PI3-K/Akt. Addition of -cyclodextrin, which depletes cholesterol from caveolae and thereby disrupts caveolar structures, was able to prevent stretch-induced assembly of PI3-K with Akt and caveolin-1 (Figure 2A). Furthermore, it prevented activation of PI3-K and phosphorylation of Akt (Figure 2B and Figure 2D). Because caveolin-1 essentially contributes to caveolae formation and serves as an adaptor molecule for multiple cell membrane molecules, involvement of caveolin-1 in stretch-induced PI3-K activation and Akt phosphorylation was examined. Incubation of cells with caveolin-1 antisense oligonucleotides (cav-1 AS ODN) but not control (reverse) ODN resulted in an almost complete abolition of caveolin-1 expression within 24 hours (Figure 2C). Incubation with cav-1 AS ODN 24 hours before stretch was able to completely prevent phosphorylation and, thereby, activation of Akt (Figure 2D).
Activation of PI3-K/Akt is essential for stretch-induced cell-cycle entry and subsequent proliferation of VSMCs.4 Consistent with experiments described earlier demonstrating a caveolin-1eCdependent activation of PI3-K/Akt during cyclic stretch, caveolin-1 AS ODN, but not control ODN, almost completely prevented stretch-induced proliferation (Figure 3A). In contrast, caveolin-1 AS ODN did not significantly alter the proliferative response of VSMCs exposed to serum stimulation (10% FCS; Figure 3B). Our data indicate that caveolin-1 mediates mechanosensitive cell cycle entry and progression of VSMCs via PI3-K/Akt.
To further substantiate the results seen by transient AS ODN-mediated caveolin-1 disruption, we isolated VSMCs from aortas of caveolin-1eC/eC mice (cav-1eC/eC). As demonstrated in rat VSMCs, exposure of mouse wild-type (WT) VSMCs to cyclic stretch for 15 minutes induced a profound activation of Akt as well as p42/44 (Erk) mitogen-activated protein kinase (MAPK) (Figure 3C). This activation was comparable to the activation seen in cells exposed to 10% FCS in the absence of cyclic stretch. Akt activation was almost abolished when VSMCs of cav-1eC/eC mice were used (Figure 3C). Although a moderate Akt activation was detected after addition of 10% FCS to cav-1eC/eC VSMCs, this activation was markedly impaired, suggesting that caveolin-1 also facilitates growth factoreCinduced Akt activation.
Similarly, Erk activation was impaired in cav-1eC/eC cells exposed to cyclic stretch, suggesting that caveolin-1 is also essential for mechanosensitive Erk activation. However, Erk was still sensitive to serum stimulation in these cells.
Twenty four hours of cyclic stretch did not trigger proliferation of cav-1eC/eC cells but of WT cells (15±1.7 versus 28±2.4 of cells in S/G2-phase, respectively; n=3, P<0.01; Figure 3D). However, the proliferative response to 10% FCS was not impaired in cav-1eC/eC VSMCs compared with their WT littermates (36.5±3.5 versus 32.1±2.8, cells in S/G2-phase; n=3, P=NS; Figure 3D). Notably, the basal proliferative rate of cav-1eC/eC cells was determined to be higher than that of WT cells (n=3, P<0.01, Figure 3D).
Cyclic Stretch Causes Activation of Src Family Kinases, Their Association With Caveolin-1 and PI3-K (p85), and Activation of PI3-K/Akt
Src nonreceptor protein tyrosine family kinases have been demonstrated to associate with caveolin-1.13 Src family kinases (SFKs) have also been shown to be involved in Akt activation.14 We examined whether the SFK c-Src, Fyn, or c-Yes associate with caveolin-1 and PI3-K (p85) under conditions of cyclic stretch. Lysates of rat VSMCs stretched for 5 minutes were immunoprecipitated with antieCc-Src, anti-Fyn, or antieCc-Yes, and an immunoblot was performed for caveolin-1. Cyclic stretch rapidly stimulated caveolin-1/Fyn, caveolin-1/c-Src, and caveolin-1/c-Yes association in VSMCs (Figure 4A). Furthermore, cyclic stretch resulted in activation of Fyn, c-Src, and c-Yes as examined by their autophosphorylation (Figure 4B). SFK activation was rapid and transient, as peak activity levels were already reached after 30 minutes of cyclic stretch (data not shown). As expected, stretch-induced activation of SFKs was prevented by the specific inhibitor, PP1. In addition, cyclic stretch resulted in a significant (4.5-fold) increase of c-Src tyrosine kinase activity (Figure 4C, n=3, P<0.01).
We subsequently examined whether SFKs are involved in stretch-induced PI3-K/Akt activation. Previously, we had shown that Akt phosphorylation, under conditions of cyclic stretch, was strictly dependent on PI3-K activation because PI3-K inhibition (LY294002 and Wortmannin) completely prevented Akt phosphorylation.4 The addition of PP1, a specific inhibitor of the Src kinase family,15 not only prevented PI3-K activation (Figure 5A) but was also able to completely prevent stretch-induced Akt phosphorylation (Figure 5B), indicating that the SFK acts upstream of PI3-K.
To further investigate the role of SFKs in PI3-K/Akt activation and to specifically probe for the involvement of c-Src in stretch-induced PI3-K activation, we transiently overexpressed a kinase-inactive mutant of c-Src [c-Src(K297R)] in rat VSMCs. Overexpression of c-Src(K297R) but not of a control vector expressing green fluorescent protein (GFP) prevented stretch-induced Akt activation (Figure 5C). The cells had been cotransfected and MACS-sorted before Western blotting to ensure analysis of a highly enriched, positively transfected cell population expressing the inactive c-Src mutant.
Integrin V3 Mediates Stretch-Induced PI3-K/Akt Activation
We previously demonstrated that mechanosensitive PI3-K activation is dependent on an intact integrin/extracellular matrix interaction,4 and V3 integrins have been shown to mediate stretch-induced VSMC proliferation.16 Therefore, experiments were performed to elucidate the role of specific integrins, in particular V3 integrins, in mechanosensitive PI3-K activation. Lysates of cells stretched for 10 minutes were immunoprecipitated with antieCPI3-K (p85), and immunoblots were performed for V and 3 integrins. Both integrin types were found to be associated with PI3-K (p85), indicating that V or 3 integrins play a role in mechanically induced PI3-K/Akt activation (Figure 6A). To further examine V3 integrin involvement in PI3-K/Akt activation, we preincubated cells with a specific V3-integrin inhibitor (XJ735). Preincubation of cells with XJ735 but not with a control peptide prevented stretch-induced Akt activation (Figure 6B), indicating that stretch-induced PI3-K activation is mediated to a large extent by V3 integrins.
Mechanosensitive Akt Activation and Neointima Formation Is Impaired in Caveolin-1eC/eC Mice
To validate the role of caveolin-1 in mechanical straineCinduced signal transduction and proliferation in vivo, jugular vein segments from cav-1eC/eC mice were exposed to arterial pressure by transplantation into the mouse common carotid artery of C57/BL6J WT recipients. After exposure of the vein grafts to arterial pressure for 15 minutes, activation (phosphorylation) of Akt and Erk was determined by immunoblotting. The results indicate that Akt activation was prevented in the veins of cav-1eC/eC mice (Figure 7A). Similarly, Erk activation was impaired in the veins of cav-1eC/eC mice compared with the veins of WT controls (Figure 7A).
To evaluate if impaired force-induced Akt (and Erk) activation of cav-1eC/eC VSMCs in vitro and in vivo finds a correlate in an altered neointima formation, vein segments were interposed into the arterial system for 6 weeks. Whereas veins of WT mice developed severe neointimal lesions, neointima formation was significantly reduced in veins derived from cav-1eC/eC mice (0.12±0.03 versus 0.19±0.03 mm2, respectively; n=8, P<0.01; Figure 7B and 7C).
Discussion
Mechanotransduction plays a critical role in vascular homeostasis. Whereas physiologically moderate cyclic stretch seems essential for maintaining vessel wall structure and for inhibition of growth factoreCstimulated proliferation of VSMCs,17 enhanced tensile force, more likely resembling pathological conditions such as those occurring in severe hypertension, venous bypass grafts, or during balloon angioplasty, has been reported to induce proliferation of VSMCs.18 In contrast to growth factoreCinduced cell cycle entry and proliferation, the mechanosensitive signaling events from the cell surface toward the cell cycle machinery are still poorly understood. Recently, we were able to demonstrate that mechanical force activates PI3-K/Akt signaling independent of growth factors. Akt in turn inactivates forkhead transcription factors, which are involved in p27Kip1 gene transactivation. Subsequent downregulation of p27Kip1 resulted in cell cycle entry and progression of VSMCs.4
In the present study, we were able to further characterize the components and mechanisms of VSMCs mechanosensing that initiate mechanically induced proliferation of VSMCs. We provide evidence that Src family kinases represent an important component of integrin-mediated PI3-K/Akt signaling and that caveolin-1 is required for efficient signaling by forming an active c-Src kinase/PI3-K/Akt module. Moreover, we demonstrate the relevance of our findings in an in vivo model of enhanced tensile force. Interposing a vein of a cav-1eC/eC mouse into the carotid artery of a WT mouse allowed us to examine the effect of force on the cav-1eC/eC vasculature independent of systemic limitations inherent in caveolin-1 gene-disrupted mice. In cav-1eC/eC veins, Akt activation was completely abolished. Nevertheless, veins of cav-1eC/eC mice still developed a neointima, although to a significantly lesser extent. Previous studies and our in vitro data may explain the in vivo results: whereas Akt was unresponsive to both serum and mechanical stimulation when caveolin-1 was deleted, exposure of these cells to serum mitogens still resulted in Erk activation. On the other hand, stretch-induced proliferation was completely prevented when VSMCs of cav-1eC/eC mice were used, whereas growth factor treatment still resulted in their cell cycle entry and progression. This points toward Akt’s predominant role for mechanically triggered proliferation. Indeed, pharmacological inhibition of Erk during stretch did not impair the proliferative response of VSMCs in vitro.4 Furthermore, our study also points toward a predominant role of caveolin-1 for mechanosensitive cell cycle entry and progression. Growth factoreCinduced proliferation, however, was not impaired when caveolin-1 was disrupted, a finding previously described.19
In vivo, the net effect of growth factors and mechanically triggered stimuli determine the amount of neointima formation. This may explain why late neointima formation was significantly reduced but not abolished in our model. Moreover, it may explain why another study that used instead an inflammatory model of neointima formation reported an increase of lesion size in cav-1eC/eC mice.20 Obviously, more factors have to be considered. In cav-1eC/eC mice, endothelial nitric oxide synthase (eNOS) is activated.21 eNOS gene transfer has been shown to prevent neointima formation in denuded rat carotid arteries.22 Therefore, augmented endothelial nitric oxide synthesis may be additionally responsible for reduced neointima formation in our in vivo model as well as reduced plaque formation in ApoEeC/eC mice interbred with cav-1eC/eC.23
As mentioned earlier, other studies have demonstrated that signaling components within caveolae may be held inactive until their activation and release by appropriate external stimuli. The p42/44 (Erk) MAP kinase cascade has been shown to be predominantly negatively regulated by caveolin.19,24 Moreover, mice lacking caveolin-1 have defects in nitric oxide and calcium signaling, and their lungs display severe abnormalities caused by uncontrolled cell proliferation and fibrosis,25 pointing toward the role of caveolin in inhibiting signaling pathways that regulate cellular proliferation in lung tissue. It is not yet clear, however, why hyperproliferative abnormalities were not found in other tissues that are normally rich in caveolin-1. This may be explained by studies demonstrating that caveolae can also stimulate signaling activity and proliferation. The association of caveolin-1 with the integrin -subunit and the tyrosine kinase Fyn, for example, leads to activation of the Ras-Erk pathway and promotes cell cycle progression.7 The PDGF and the insulin receptors also seem to initiate their signal transduction from caveolae.26 Our data support the hypothesis that in the vasculature, which is constantly exposed to alternating mechanical force and different growth factors, a dual role of caveolin-1 may contribute to a sensitive balance of anti- and pro-proliferative effects that allow the vessel to adapt to acute or chronic changes. Interestingly, a dual role of caveolin-1 toward eNOS has recently been demonstrated: although caveolin-1 repressed basal eNOS activity, it was crucial for agonist- (vascular endothelial growth factoreC) induced eNOS activation.27
In this study, we also demonstrate that cholesterol depletion of the plasma membrane by -cyclodextrin prevents PI3-K/Akt activation. Caveolae form from cholesterol- and sphingolipid-rich rafts in the membrane in a process that requires caveolin-1. Furthermore, the function of caveolae is dependent on a sufficient level of cholesterol in the plasma membrane, and caveolin-1 is involved in maintaining caveolar cholesterol levels.28 Consistent with our results, reduction of plasma membrane cholesterol levels with cholesterol-binding agents has been shown to inhibit specific agonist-stimulated signaling pathways, eg, insulin-stimulated insulin receptor substrate-1 phosphorylation29 or shear stress,30 and nerve growth factoreCinduced31 and endothelin-1eCinduced32 activation of the extracellular signal-regulated kinase, Erk. Although our data suggest that caveolin-1 acts directly through protein-protein interactions, it may also operate indirectly by maintaining caveolar structure and integrity necessary for mechanosensitive proliferation through integrin/c-Src/PI3-K/Akt interaction.
Immunocytochemical staining of VSMCs indicates a force-dependent recruitment of PI3-K and caveolin-1 to focal adhesion sites, suggesting that PI3-K plays a role in integrin-mediated cellular response to mechanical stimuli. It has been shown that integrins are, indeed, sequestered into caveolar microdomains of the plasma membrane,7,33 implying a requirement of caveolar microdomains for integrating integrin-mediated mechanotransduction toward an intracellular signal. In VSMCs, the proliferative response to strain was abrogated by antibodies to both V and 3 integrins, but not 1 integrins.16,34 In accordance with these findings, we find a dependency of tensile stress-mediated PI3-K/Akt activation on V3 integrins. Furthermore, Src family kinases are required for the force-dependent formation of focal adhesion complexes and strengthening of V3 integrin-cytoskeleton connections.35 In addition, an involvement of Src family kinases in PI3-K and in Akt activation has been reported before.36 In this study, we demonstrate that a pharmacological inhibitor of the Src kinase family tyrosine kinases (PP1) prevents not only Akt activation and proliferation, but also PI3-K activation, indicating that mechanosensitive c-Src kinase signaling lies upstream of PI3-K. Our findings support the concept that c-Src is an additional, essential component for mechanosensitive PI3-K activation.
Mechanosensing of VSMCs via V3 integrins/caveolin-1/c-Src kinaseeCdependent activation of PI3-K/Akt signaling and subsequent cellular proliferation may play an important role in physiological vascular remodeling processes and the pathophysiology of vascular proliferative diseases. Our data further add to the understanding of mechanisms involved in vascular homeostasis and the pathophysiology of proliferative disease processes and may have novel implications for the future design of therapeutic interventions.
Acknowledgments
R.C.B.-D. is supported by the Deutsche Forschungsgemeinschaft (DFG, BR 1603/4-1). D.G.S. is scholar of the Deutsche Gesellschaft fe Kardiologie (German Cardiac Society research scholarship).
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Peiro S, Comella JX, Enrich C, Martin-Zanca D, Rocamora N. PC12 cells have caveolae that contain TrkA. Caveolae-disrupting drugs inhibit nerve growth factor-induced, but not epidermal growth factor-induced, MAPK phosphorylation. J Biol Chem. 2000; 275: 37846eC37852.
Teixeira A, Chaverot N, Schroder C, Strosberg AD, Couraud PO, Cazaubon S. Requirement of caveolae microdomains in extracellular signal-regulated kinase and focal adhesion kinase activation induced by endothelin-1 in primary astrocytes. J Neurochem. 1999; 72: 120eC128.
Chapman HA, Wei Y, Simon DI, Waltz DA. Role of urokinase receptor and caveolin in regulation of integrin signaling. Thromb Haemost. 1999; 82: 291eC297.
Sudhir K, Wilson E, Chatterjee K, Ives HE. Mechanical strain and collagen potentiate mitogenic activity of angiotensin II in rat vascular smooth muscle cells. J Clin Invest. 1993; 92: 3003eC3007.
von Wichert G, Jiang G, Kostic A, De Vos K, Sap J, Sheetz MP. RPTP-alpha acts as a transducer of mechanical force on alphav/beta3-integrin-cytoskeleton linkages. J Cell Biol. 2003; 161: 143eC153.
Jiang T, Qiu Y. Interaction between Src and a C-terminal proline-rich motif of Akt is required for Akt activation. J Biol Chem. 2003; 278: 15789eC15793.(Daniel G. Sedding, Jennif)
Institute for Anatomy and Cell Biology (W.K.), Giessen University, Giessen, Germany
the Department of Internal Medicine II/Cardiology (C.S., R.H.S., R.C.B.-D.), Dresden University of Technology, Dresden, Germany.
Abstract
Mechanotransduction represents an integral part of vascular homeostasis and contributes to vascular lesion formation. Previously, we demonstrated a mechanosensitive activation of phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt) resulting in p27Kip1 transcriptional downregulation and cell cycle entry of vascular smooth muscle cells (VSMC). In this study, we further elucidated the signaling from outside-in toward PI3-K/Akt in vitro and in an in vivo model of elevated tensile force. When VSMC were subjected to cyclic stretch (0.5 Hz at 125% resting length), PI3-K, Akt, and Src kinases were found activated. Disrupting caveolar structures with -cyclodextrin or transfection of VSMC with caveolin-1 antisense oligonucleotides (ODN) prevented PI3-K and Akt activation and cell cycle entry. Furthermore, PI3-K and Akt were resistant to activation when Src kinases were inhibited pharmacologically or by overexpression of a kinase-dead c-Src mutant. V3 integrins were identified to colocalize with PI3-K/caveolin-1 complexes, and blockade of V3 integrins prevented Akt activation. The central role of caveolin-1 in mechanotransduction was further examined in an in vivo model of elevated tensile force. Interposition of wild-type (WT) jugular veins into WT carotid arteries resulted in a rapid Akt activation within the veins that was almost abolished when veins of caveolin-1 knockout (KO) mice were used. Furthermore, late neointima formation within the KO veins was significantly reduced. Our study provides evidence that PI3-K/Akt is critically involved in mechanotransduction of VSMC in vitro and within the vasculature in vivo. Furthermore, caveolin-1 is essential for the integrin-mediated activation of PI3-K/Akt.
Key Words: remodeling muscle, smooth signal transduction stress vasculature
Introduction
It is currently recognized that the machinery governing the cell cycle regulates multiple cellular functions in the cardiovascular system, thereby maintaining the homeostasis of the vasculature and allowing its adaptation to acute and chronic changes. Besides organizing cellular proliferation, the cell cycle is involved in migration, apoptosis, and hypertrophy.1
One of the major constituents of the blood vessel wall responsible for the maintenance of vessel structures and functions are vascular smooth muscle cells (VSMCs). In the vasculature, VSMCs are constantly exposed to alternating mechanical forces. Under normal tensile stress, VSMCs are relatively insensitive to mitogens. During altered mechanical stress (eg, high blood pressure), however, VSMCs upregulate protein synthesis in response to growth factors, dedifferentiate, and increase their proliferative rate, resulting in medial hypertrophy and intimal hyperplasia.2 Whereas the commonly accepted "response to injury" hypothesis suggests that growth factors are locally released, thereby initiating cell cycle entry and progression of vascular cells, the signaling pathways arising solely from mechanical force have just partially come to light.3 Recently, we demonstrated that the earliest cell cycle events can occur in a mechanosensitive fashion independently of newly released or synthesized growth factors but that they are dependent on an intact integrin signaling.4
Besides mediating cell adhesion, integrins transmit extracellular stimuli into intracellular signaling events.5 Thus, integrins mediate VSMC dedifferentiation, migration, proliferation, and apoptosis.6 Signaling through integrins requires physical interaction with other membrane proteins and subsequent association with signal transduction proteins of the cytoplasm. Caveolin-1 functions as a membrane adaptor. For example, caveolin-1 links the integrin -subunit to the c-Src kinase pathway and subsequently to the mitogen-activated protein kinase pathway (MAPK) to promote cell cycle progression.7 Another pathway critical for VSMC proliferation consists of the phosphoinositide 3-kinase(PI3-K)/protein kinase B (Akt) signal transduction pathway.8 Although the MAPK pathway had previously been shown to be responsive to mechanical force,9 we were recently able to demonstrate that cyclic stretch resulted in a rapid PI3-K/Akt activation as well, thereby inducing forkhead transcription factoreCdependent downregulation of the cell cycle inhibitor p27Kip1.4 This chain of events facilitated mechanosensitive cell cycle entry and proliferation of VSMCs in contrast to the posttranscriptional downregulation of p27Kip1 protein in VSMCs stimulated with serum mitogens. In this way, p27Kip1 may serve as a differential sensor for growth factoreC and mechanically induced cell cycle entry in VSMCs. In the present study, we further elucidated the integrin/adaptor protein interactions resulting in growth factoreCindependent PI3-K/Akt activation and subsequent cell cycle entry. We present evidence that the structural protein caveolin-1 is essential for integrin-mediated PI3-K/Akt activation during cyclic stretch of VSMCs in vitro and in an in vivo model of elevated tensile force. In our in vitro model, mechanical force induces the formation of functional signaling complexes composed of V3 integrins, caveolin-1, PI3-K/Akt, and the nonreceptor tyrosine kinase c-Src, resulting in PI3-K/Akt activation and thereby facilitating a mechanosentive proliferative response of VSMCs.
Materials and Methods
Cell Culture, Stretch Apparatus, and Experimental Conditions
Primary cultures of VSMCs were initiated by enzymatic dissociation from the aorta of 7- to 8-week-old male Sprague-Dawley rats (Charles River Breeding Laboratories, Kingston, NY).10 The cells were seeded (10 000 cells/cm2) onto 6-well fibronectin-coated FlexI plates (Flexcell). Studies were conducted on VSMCs (passages 7 to 12) after achieving confluence in 10% FCS/DMEM/F12, followed by serum withdrawal for 2 days to achieve quiescence. On the day of the experiment, fresh serum-free medium was substituted and uniaxial cyclic stretch was applied with a flexercell apparatus (125% resting length, 0.5 Hz) in a tissue culture incubator.
Immunocytochemistry
VSMCs were grown on fibronectin-coated flexercell wells. Quiescent or stretched (30 minutes) cells were fixed for 10 minutes in ice-cold acetone. Cells were covered for 20 minutes with 10% normal goat serum, followed by incubation with rabbit anti-caveolin-1 (1:100) and mouse anti PI3-K (p85, 1:50) for 1 hour in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin. After two washing steps (10 minutes in PBS), cells were incubated with secondary antibodies for 40 minutes: donkey anti-rabbit IgG conjugated to FITC (1:200) and goat anti-mouse IgG conjugated to Alexa Fluor 546 (1:200). After washing, cells were mounted in Vectashield mounting medium H-1000 containing DAPI (5 e/mL, Linaris) and evaluated using an epifluorescence microscope (DMRB, Leica). Negative controls were performed using only the secondary antibody.
Preparation of Cellular Lysates and Immunoblot Analysis
Specific protein content in cell lysates was analyzed by immunoblot as previously described.11 Briefly, cell lysis buffer contained 50 mmol/L HEPES pH 7.4, 100 mmol/L NaCl, 50 mmol/L NaF, 5 mmol/L glycerophosphate, 2 mmol/L EDTA, 2 mmol/L EGTA, 1 mmol/L vanadat, 0.5% octyglucopyranoside, PMSF 100 e/mL, aprotinin 30 e蘈/mL, leupeptin 1 ng/mL, and okadaic acid 10 eol/L. Lysate proteins (20 e) were run on a polyacrylamide gel and blotted onto nitrocellulose (Hybond-ECL, Amersham). After blocking, blots were incubated with primary antibody for 1 hour at room temperature. Specific proteins were detected by enhanced chemiluminescence (ECL+, Amersham) after labeling with horseradish peroxidase-labeled secondary antibody (1:2000 for 1 hour) according to the manufacturer’s instructions.
Flow Cytometry
Cells were harvested by trypsinization, fixed overnight with 75% methanol, washed, and incubated with 100 e/mL RNase (Oncogene Research Products) and 10 e/mL propidium iodide in PBS for 1 hour at 37°C. Samples were analyzed for DNA content using a high-speed cell sorter (EPICs Altra, Beckman Coulter). Data were computer-analyzed with commercially available software (Multicycle, Phoenix Flow Systems).
Mice and Vein Graft Procedure
Caveolin-1eC/eC mice were provided by M. Drab, Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany. All animal experiments were performed according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals (Giessen University). The vein graft procedure has been described previously.12 Briefly, mice were anesthetized with ketamine (50 mg/kg body weight, IP). The jugular vein was harvested from the donor. In the recipient, the left common carotid artery was mobilized from proximal to the distal bifurcation, cut in the middle, and a cuff (0.63 mm outside diameter and 0.5 mm inside diameter, Portex LTD) placed at each end. At each end, the artery was turned inside out over the cuff and ligated. The vein segment was grafted between the two ends of the carotid artery by sleeving the ends of the vein over the artery-cuff and ligating them together with an 8-0 suture.
For Western blot experiments, vein grafts were perfused with PBS, excised immediately, and snap-frozen in liquid nitrogen until further use.
Statistical Analysis
Data are given as mean±SEM. Statistical analysis was performed by ANOVA. Post hoc analysis was performed by the method of Bonferroni. All experiments, including the immunoblots, were independently repeated at least three times.
For detailed description of reagents, transfection procedures, magnetic activated cell sorting (MACS), phosphoinositide 3-kinase assay, Src family kinase assay, and histological evaluation of tissue sections please refer to the online data supplement available at http://circres.ahajournals.org.
Results
Mechanical Strain Induces Colocalization of PI3-K With Caveolin-1 and Their Clustering at Focal Adhesion Sites
Previously, we had demonstrated that stretch-induced cell cycle entry and progression can be prevented by pharmacological inhibition of PI3-K (LY294002 and wortmannin) or overexpression of a constitutive negative Akt, indicating a requirement for PI3-K/Akt in mechanically induced proliferation of VSMCs.4 In the present examination of cellular distribution, it was found that in quiescent cells, PI3-K had a homogeneous cytosolic distribution and that caveolin-1 was distributed at the cell surface in a diffuse, punctuate manner (Figure 1A and 1B). After 15 minutes of cyclic stretch, PI3-K and caveolin-1 accumulated in clusters at the cell membrane. Double-staining suggested their colocalization (Figure 1A and 1B). The PI3-K/caveolin-1 complexes seemed to be located at focal adhesion sites as implicated by their colocalization with vinculin (shown for caveolin-1; Figure 1A and 1B).
Mechanosensitive Activation of PI3-K/Akt Requires Intact Caveolar Structures and Caveolin-1
We examined whether caveolar structures are required for the mechanosensitive activation of PI3-K/Akt. Addition of -cyclodextrin, which depletes cholesterol from caveolae and thereby disrupts caveolar structures, was able to prevent stretch-induced assembly of PI3-K with Akt and caveolin-1 (Figure 2A). Furthermore, it prevented activation of PI3-K and phosphorylation of Akt (Figure 2B and Figure 2D). Because caveolin-1 essentially contributes to caveolae formation and serves as an adaptor molecule for multiple cell membrane molecules, involvement of caveolin-1 in stretch-induced PI3-K activation and Akt phosphorylation was examined. Incubation of cells with caveolin-1 antisense oligonucleotides (cav-1 AS ODN) but not control (reverse) ODN resulted in an almost complete abolition of caveolin-1 expression within 24 hours (Figure 2C). Incubation with cav-1 AS ODN 24 hours before stretch was able to completely prevent phosphorylation and, thereby, activation of Akt (Figure 2D).
Activation of PI3-K/Akt is essential for stretch-induced cell-cycle entry and subsequent proliferation of VSMCs.4 Consistent with experiments described earlier demonstrating a caveolin-1eCdependent activation of PI3-K/Akt during cyclic stretch, caveolin-1 AS ODN, but not control ODN, almost completely prevented stretch-induced proliferation (Figure 3A). In contrast, caveolin-1 AS ODN did not significantly alter the proliferative response of VSMCs exposed to serum stimulation (10% FCS; Figure 3B). Our data indicate that caveolin-1 mediates mechanosensitive cell cycle entry and progression of VSMCs via PI3-K/Akt.
To further substantiate the results seen by transient AS ODN-mediated caveolin-1 disruption, we isolated VSMCs from aortas of caveolin-1eC/eC mice (cav-1eC/eC). As demonstrated in rat VSMCs, exposure of mouse wild-type (WT) VSMCs to cyclic stretch for 15 minutes induced a profound activation of Akt as well as p42/44 (Erk) mitogen-activated protein kinase (MAPK) (Figure 3C). This activation was comparable to the activation seen in cells exposed to 10% FCS in the absence of cyclic stretch. Akt activation was almost abolished when VSMCs of cav-1eC/eC mice were used (Figure 3C). Although a moderate Akt activation was detected after addition of 10% FCS to cav-1eC/eC VSMCs, this activation was markedly impaired, suggesting that caveolin-1 also facilitates growth factoreCinduced Akt activation.
Similarly, Erk activation was impaired in cav-1eC/eC cells exposed to cyclic stretch, suggesting that caveolin-1 is also essential for mechanosensitive Erk activation. However, Erk was still sensitive to serum stimulation in these cells.
Twenty four hours of cyclic stretch did not trigger proliferation of cav-1eC/eC cells but of WT cells (15±1.7 versus 28±2.4 of cells in S/G2-phase, respectively; n=3, P<0.01; Figure 3D). However, the proliferative response to 10% FCS was not impaired in cav-1eC/eC VSMCs compared with their WT littermates (36.5±3.5 versus 32.1±2.8, cells in S/G2-phase; n=3, P=NS; Figure 3D). Notably, the basal proliferative rate of cav-1eC/eC cells was determined to be higher than that of WT cells (n=3, P<0.01, Figure 3D).
Cyclic Stretch Causes Activation of Src Family Kinases, Their Association With Caveolin-1 and PI3-K (p85), and Activation of PI3-K/Akt
Src nonreceptor protein tyrosine family kinases have been demonstrated to associate with caveolin-1.13 Src family kinases (SFKs) have also been shown to be involved in Akt activation.14 We examined whether the SFK c-Src, Fyn, or c-Yes associate with caveolin-1 and PI3-K (p85) under conditions of cyclic stretch. Lysates of rat VSMCs stretched for 5 minutes were immunoprecipitated with antieCc-Src, anti-Fyn, or antieCc-Yes, and an immunoblot was performed for caveolin-1. Cyclic stretch rapidly stimulated caveolin-1/Fyn, caveolin-1/c-Src, and caveolin-1/c-Yes association in VSMCs (Figure 4A). Furthermore, cyclic stretch resulted in activation of Fyn, c-Src, and c-Yes as examined by their autophosphorylation (Figure 4B). SFK activation was rapid and transient, as peak activity levels were already reached after 30 minutes of cyclic stretch (data not shown). As expected, stretch-induced activation of SFKs was prevented by the specific inhibitor, PP1. In addition, cyclic stretch resulted in a significant (4.5-fold) increase of c-Src tyrosine kinase activity (Figure 4C, n=3, P<0.01).
We subsequently examined whether SFKs are involved in stretch-induced PI3-K/Akt activation. Previously, we had shown that Akt phosphorylation, under conditions of cyclic stretch, was strictly dependent on PI3-K activation because PI3-K inhibition (LY294002 and Wortmannin) completely prevented Akt phosphorylation.4 The addition of PP1, a specific inhibitor of the Src kinase family,15 not only prevented PI3-K activation (Figure 5A) but was also able to completely prevent stretch-induced Akt phosphorylation (Figure 5B), indicating that the SFK acts upstream of PI3-K.
To further investigate the role of SFKs in PI3-K/Akt activation and to specifically probe for the involvement of c-Src in stretch-induced PI3-K activation, we transiently overexpressed a kinase-inactive mutant of c-Src [c-Src(K297R)] in rat VSMCs. Overexpression of c-Src(K297R) but not of a control vector expressing green fluorescent protein (GFP) prevented stretch-induced Akt activation (Figure 5C). The cells had been cotransfected and MACS-sorted before Western blotting to ensure analysis of a highly enriched, positively transfected cell population expressing the inactive c-Src mutant.
Integrin V3 Mediates Stretch-Induced PI3-K/Akt Activation
We previously demonstrated that mechanosensitive PI3-K activation is dependent on an intact integrin/extracellular matrix interaction,4 and V3 integrins have been shown to mediate stretch-induced VSMC proliferation.16 Therefore, experiments were performed to elucidate the role of specific integrins, in particular V3 integrins, in mechanosensitive PI3-K activation. Lysates of cells stretched for 10 minutes were immunoprecipitated with antieCPI3-K (p85), and immunoblots were performed for V and 3 integrins. Both integrin types were found to be associated with PI3-K (p85), indicating that V or 3 integrins play a role in mechanically induced PI3-K/Akt activation (Figure 6A). To further examine V3 integrin involvement in PI3-K/Akt activation, we preincubated cells with a specific V3-integrin inhibitor (XJ735). Preincubation of cells with XJ735 but not with a control peptide prevented stretch-induced Akt activation (Figure 6B), indicating that stretch-induced PI3-K activation is mediated to a large extent by V3 integrins.
Mechanosensitive Akt Activation and Neointima Formation Is Impaired in Caveolin-1eC/eC Mice
To validate the role of caveolin-1 in mechanical straineCinduced signal transduction and proliferation in vivo, jugular vein segments from cav-1eC/eC mice were exposed to arterial pressure by transplantation into the mouse common carotid artery of C57/BL6J WT recipients. After exposure of the vein grafts to arterial pressure for 15 minutes, activation (phosphorylation) of Akt and Erk was determined by immunoblotting. The results indicate that Akt activation was prevented in the veins of cav-1eC/eC mice (Figure 7A). Similarly, Erk activation was impaired in the veins of cav-1eC/eC mice compared with the veins of WT controls (Figure 7A).
To evaluate if impaired force-induced Akt (and Erk) activation of cav-1eC/eC VSMCs in vitro and in vivo finds a correlate in an altered neointima formation, vein segments were interposed into the arterial system for 6 weeks. Whereas veins of WT mice developed severe neointimal lesions, neointima formation was significantly reduced in veins derived from cav-1eC/eC mice (0.12±0.03 versus 0.19±0.03 mm2, respectively; n=8, P<0.01; Figure 7B and 7C).
Discussion
Mechanotransduction plays a critical role in vascular homeostasis. Whereas physiologically moderate cyclic stretch seems essential for maintaining vessel wall structure and for inhibition of growth factoreCstimulated proliferation of VSMCs,17 enhanced tensile force, more likely resembling pathological conditions such as those occurring in severe hypertension, venous bypass grafts, or during balloon angioplasty, has been reported to induce proliferation of VSMCs.18 In contrast to growth factoreCinduced cell cycle entry and proliferation, the mechanosensitive signaling events from the cell surface toward the cell cycle machinery are still poorly understood. Recently, we were able to demonstrate that mechanical force activates PI3-K/Akt signaling independent of growth factors. Akt in turn inactivates forkhead transcription factors, which are involved in p27Kip1 gene transactivation. Subsequent downregulation of p27Kip1 resulted in cell cycle entry and progression of VSMCs.4
In the present study, we were able to further characterize the components and mechanisms of VSMCs mechanosensing that initiate mechanically induced proliferation of VSMCs. We provide evidence that Src family kinases represent an important component of integrin-mediated PI3-K/Akt signaling and that caveolin-1 is required for efficient signaling by forming an active c-Src kinase/PI3-K/Akt module. Moreover, we demonstrate the relevance of our findings in an in vivo model of enhanced tensile force. Interposing a vein of a cav-1eC/eC mouse into the carotid artery of a WT mouse allowed us to examine the effect of force on the cav-1eC/eC vasculature independent of systemic limitations inherent in caveolin-1 gene-disrupted mice. In cav-1eC/eC veins, Akt activation was completely abolished. Nevertheless, veins of cav-1eC/eC mice still developed a neointima, although to a significantly lesser extent. Previous studies and our in vitro data may explain the in vivo results: whereas Akt was unresponsive to both serum and mechanical stimulation when caveolin-1 was deleted, exposure of these cells to serum mitogens still resulted in Erk activation. On the other hand, stretch-induced proliferation was completely prevented when VSMCs of cav-1eC/eC mice were used, whereas growth factor treatment still resulted in their cell cycle entry and progression. This points toward Akt’s predominant role for mechanically triggered proliferation. Indeed, pharmacological inhibition of Erk during stretch did not impair the proliferative response of VSMCs in vitro.4 Furthermore, our study also points toward a predominant role of caveolin-1 for mechanosensitive cell cycle entry and progression. Growth factoreCinduced proliferation, however, was not impaired when caveolin-1 was disrupted, a finding previously described.19
In vivo, the net effect of growth factors and mechanically triggered stimuli determine the amount of neointima formation. This may explain why late neointima formation was significantly reduced but not abolished in our model. Moreover, it may explain why another study that used instead an inflammatory model of neointima formation reported an increase of lesion size in cav-1eC/eC mice.20 Obviously, more factors have to be considered. In cav-1eC/eC mice, endothelial nitric oxide synthase (eNOS) is activated.21 eNOS gene transfer has been shown to prevent neointima formation in denuded rat carotid arteries.22 Therefore, augmented endothelial nitric oxide synthesis may be additionally responsible for reduced neointima formation in our in vivo model as well as reduced plaque formation in ApoEeC/eC mice interbred with cav-1eC/eC.23
As mentioned earlier, other studies have demonstrated that signaling components within caveolae may be held inactive until their activation and release by appropriate external stimuli. The p42/44 (Erk) MAP kinase cascade has been shown to be predominantly negatively regulated by caveolin.19,24 Moreover, mice lacking caveolin-1 have defects in nitric oxide and calcium signaling, and their lungs display severe abnormalities caused by uncontrolled cell proliferation and fibrosis,25 pointing toward the role of caveolin in inhibiting signaling pathways that regulate cellular proliferation in lung tissue. It is not yet clear, however, why hyperproliferative abnormalities were not found in other tissues that are normally rich in caveolin-1. This may be explained by studies demonstrating that caveolae can also stimulate signaling activity and proliferation. The association of caveolin-1 with the integrin -subunit and the tyrosine kinase Fyn, for example, leads to activation of the Ras-Erk pathway and promotes cell cycle progression.7 The PDGF and the insulin receptors also seem to initiate their signal transduction from caveolae.26 Our data support the hypothesis that in the vasculature, which is constantly exposed to alternating mechanical force and different growth factors, a dual role of caveolin-1 may contribute to a sensitive balance of anti- and pro-proliferative effects that allow the vessel to adapt to acute or chronic changes. Interestingly, a dual role of caveolin-1 toward eNOS has recently been demonstrated: although caveolin-1 repressed basal eNOS activity, it was crucial for agonist- (vascular endothelial growth factoreC) induced eNOS activation.27
In this study, we also demonstrate that cholesterol depletion of the plasma membrane by -cyclodextrin prevents PI3-K/Akt activation. Caveolae form from cholesterol- and sphingolipid-rich rafts in the membrane in a process that requires caveolin-1. Furthermore, the function of caveolae is dependent on a sufficient level of cholesterol in the plasma membrane, and caveolin-1 is involved in maintaining caveolar cholesterol levels.28 Consistent with our results, reduction of plasma membrane cholesterol levels with cholesterol-binding agents has been shown to inhibit specific agonist-stimulated signaling pathways, eg, insulin-stimulated insulin receptor substrate-1 phosphorylation29 or shear stress,30 and nerve growth factoreCinduced31 and endothelin-1eCinduced32 activation of the extracellular signal-regulated kinase, Erk. Although our data suggest that caveolin-1 acts directly through protein-protein interactions, it may also operate indirectly by maintaining caveolar structure and integrity necessary for mechanosensitive proliferation through integrin/c-Src/PI3-K/Akt interaction.
Immunocytochemical staining of VSMCs indicates a force-dependent recruitment of PI3-K and caveolin-1 to focal adhesion sites, suggesting that PI3-K plays a role in integrin-mediated cellular response to mechanical stimuli. It has been shown that integrins are, indeed, sequestered into caveolar microdomains of the plasma membrane,7,33 implying a requirement of caveolar microdomains for integrating integrin-mediated mechanotransduction toward an intracellular signal. In VSMCs, the proliferative response to strain was abrogated by antibodies to both V and 3 integrins, but not 1 integrins.16,34 In accordance with these findings, we find a dependency of tensile stress-mediated PI3-K/Akt activation on V3 integrins. Furthermore, Src family kinases are required for the force-dependent formation of focal adhesion complexes and strengthening of V3 integrin-cytoskeleton connections.35 In addition, an involvement of Src family kinases in PI3-K and in Akt activation has been reported before.36 In this study, we demonstrate that a pharmacological inhibitor of the Src kinase family tyrosine kinases (PP1) prevents not only Akt activation and proliferation, but also PI3-K activation, indicating that mechanosensitive c-Src kinase signaling lies upstream of PI3-K. Our findings support the concept that c-Src is an additional, essential component for mechanosensitive PI3-K activation.
Mechanosensing of VSMCs via V3 integrins/caveolin-1/c-Src kinaseeCdependent activation of PI3-K/Akt signaling and subsequent cellular proliferation may play an important role in physiological vascular remodeling processes and the pathophysiology of vascular proliferative diseases. Our data further add to the understanding of mechanisms involved in vascular homeostasis and the pathophysiology of proliferative disease processes and may have novel implications for the future design of therapeutic interventions.
Acknowledgments
R.C.B.-D. is supported by the Deutsche Forschungsgemeinschaft (DFG, BR 1603/4-1). D.G.S. is scholar of the Deutsche Gesellschaft fe Kardiologie (German Cardiac Society research scholarship).
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