Depletion of plasma membrane cholesterol dampens hydrostatic pressure and shear stress-induced mechanotransduction pathways in osteoblast cultures

Jeffrey T. Ferraro,1 Mani Daneshmand,1 Rena Bizios,2 and Victor Rizzo1

1Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208; and 2Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180

Submitted 30 May 2003 ; accepted in final form 20 November 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The preferential association of cholesterol and sphingolipids within plasma membranes forms organized compartments termed lipid rafts. Addition of caveolin proteins to this lipid milieu induces the formation of specialized invaginated plasma membrane structures called caveolae. Both lipid rafts and caveolae are purported to function in vesicular transport and cell signaling. We and others have shown that disassembly of rafts and caveolae through depletion of plasma membrane cholesterol mitigates mechanotransduction processes in endothelial cells. Because osteoblasts are subjected to fluid-mechanical forces, we hypothesize that cholesterol-rich plasma membrane microdomains also serve the mechanotransduction process in this cell type. Cultured human fetal osteoblasts were subjected to either sustained hydrostatic pressure or laminar shear stress using a pressure column or parallel-plate apparatus, respectively. We found that sustained hydrostatic pressure induced protein tyrosine phosphorylation, activation of extracellular signal-regulated kinase (ERK)1/2, and enhanced expression of c-fos in both time- and magnitude-dependent manners. Similar responses were observed in cells subjected to laminar shear stress. Both sustained hydrostatic pressure- and shear stress-induced signaling were significantly reduced in osteoblasts pre-exposed to either filipin or methyl-{beta}-cyclodextrin. These mechanotransduction responses were restored on reconstitution of lipid rafts and caveolae, which suggests that cholesterol-rich plasma membrane microdomains participate in the mechanotransduction process in osteoblasts. In addition, mechanical force-induced phosphoproteins were localized within caveolin-containing membranes. These data support the concept that lipid rafts and caveolae serve a general function as cell surface mechanotransduction sites within the plasma membrane.

lipid rafts; caveolae; extracellular signal-regulated kinase


BIOMECHANICAL STRESSES GENERATED in response to mechanical loading of the skeletal system play an important role in bone development and homeostasis; removal of physical stimuli proves detrimental to the skeletal tissue in vivo (17, 51). Osteoblasts are primary regulators of bone metabolism and appear to be particularly sensitive to changes in their physical environment. In response to fluid flow-induced shear stress, osteoblasts generate (5, 2427, 39) prostaglandin E2 (PGE2), inositol 1,4,5-trisphosphate (IP3), cAMP, cyclooxygenase-2 (COX-2), and nitric oxide (NO). Likewise, application of compressive forces to osteoblast cultures stimulates prostacyclin production and enhances expression of the early response gene c-fos (9) and alkaline phosphatase (19). Although the effects of mechanical stimulation on osteoblast function pertinent to new bone formation continue to be the center of study, the basic mechanisms by which biomechanical forces are detected and translated into biological signals within these cells are not clearly defined.

There is accumulating evidence to support the concept that mechanochemical signaling occurs in spatially discrete sub-compartments within cells. Perhaps the best characterized of these are the adhesions and focal contacts present on cell membranes. In endothelial cells, shear stress has been shown to activate integrin complexes that trigger localized accumulation of a wide variety of signaling molecules (2, 18, 48). Similar evidence for integrin-mediated mechanotransduction in osteoblasts exposed to shear stress has been observed, suggesting that focal contacts may serve as mechanosensitive sites within this cell type (22).

The recent discovery that many receptors and signaling molecules preferentially localize to plasma membrane regions composed of glycerolphospholipids and enriched in cholesterol and sphingolipids, termed lipid rafts and caveolae, has led to the hypothesis that these membrane microdomains also serve as signaling compartments (4, 38, 40). The importance of plasma membrane microdomain signaling was highlighted in a recent study that identified caveolin-enriched membrane fractions as important structures necessary for proper growth factor-mediated signaling in osteoblasts (43). Interestingly, mechanotransduction in osteoblasts appears to involve many of the same signaling components that are necessary for growth-factor signaling. On the basis of these observations and our past studies demonstrating that acute mechanosignaling events are localized within and depend on the structural integrity of caveolae in endothelial cells (28, 30), we tested whether cholesterol-rich caveolin-containing plasma membrane domains are necessary for efficient mechanotransduction in cultured osteoblasts.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. Human fetal osteoblasts (hFOB 1.19, American Type Culture Collection) were cultured in a 1:1 mixture of Ham's F-12 medium/DMEM without phenol red and supplemented with 10% FBS. Cells were cultured in a humidified 5% CO2-95% air environment at 37°C.

Hydrostatic pressure experiments. Osteoblasts were seeded onto 35-mm round glass coverslips and cultured to confluency. Before placement in the hydrostatic pressure chamber, cells were deprived of serum for 2 h to reduce the level of growth factor-stimulated signaling and proliferation. Coverslips were fixed to the base of the sterile polypropylene cylinder (height, 11 cm; diameter, 3 cm) with a small amount of sterile vacuum grease placed on the side opposite of the cells. Osteoblasts were then exposed to sustained hydrostatic pressures of either 5 or 10 mmHg above ambient atmospheric pressure for either 10 or 30 min. In some experiments, osteoblasts were pretreated with methyl-{beta}-cyclodextrin (CD) or filipin as described (see Lipid raft and caveolae manipulation with cholesterol-interacting agents). After pressure exposure, the osteoblasts were lysed in 1 ml of HEPES-buffered sucrose that contained protease inhibitors [0.25 M sucrose, 25 mM HEPES at pH 7, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µg/ml o-phenanthroline, 10 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, and 50 µg/ml trans-epoxysuccinyl-L-leucinamido(4-guanidino)butane] and were processed for SDS-PAGE and Western blot analysis.

Shear-stress experiments. Osteoblasts were seeded onto 75 x 38-mm glass slides and cultured to confluency. After serum deprivation for 2 h, cells were subjected to 10 dyn/cm2 of laminar shear stress in a parallel-plate apparatus for 10 min. Serum-free culture medium was recirculated using a flow circuit composed of a variable-speed peristaltic pump, a fluid capacitor that damped pulsation, and a reservoir with culture medium. Laminar shear stress was achieved by circulating media through a flow chamber created by separating the glass slide and a Plexiglas top plate with a 12-µm silicon gasket. Temperature was maintained at 37°C, and pH and oxygen levels were controlled with a humidified 95% air-5% CO2 gas mixture flowing over the medium in the reservoir. Shear stress was quantified using the following equation: shear stress = 6µ/wh2, where µ is fluid viscosity, is the volumetric flow rate, and w and h are the chamber width and height, respectively. In some experiments, osteobalsts were incubated with CD during the final 30 min of the serum-deprivation period (as described in Lipid raft and caveolae manipulation with cholesterol-interacting agents).

Lipid raft and caveolae manipulation with cholesterol-interacting agents. Cholesterol is an essential component of lipid rafts and caveolae and is required for the structural integrity of these microdomains. CD is a cyclic oligosaccharide that effectively extracts cholesterol from the plasma membrane, which results in lipid raft and caveolae disassembly (4, 38). To evaluate the role of cholesterol on mechanotransduction processes, osteoblasts were incubated in serum-free media that contained 1 mM CD for 30 min at 37°C before exposure to either hydrostatic pressure or shear stress. In a separate set of experiments, cholesterol was added back to cholesterol-depleted osteoblast cell cultures to reconstitute disassembled rafts. Briefly, repletion with cholesterol was accomplished by incubating cells in the presence of a cholesterol-CD mixture for 1 h at 37°C. A stock solution of 0.4 mg/ml cholesterol and 10% CD was prepared by vortexing at 40°C in 10 ml of 10% CD with 200 µl of cholesterol (20 mg/ml in ethanol solution; Ref. 20).

Filipin is a polyene macrolide antibiotic. This class of drugs binds sterols such as cholesterol and can cause reversible disassembly of rafts and caveolae (33, 37). As a complementary set of experiments to the CD studies, cells were pretreated with 5 µg/ml filipin for 5 min at 37°C before pressure treatment.

Preparation of caveolin-containing lipid raft membranes. Caveolin-enriched, light-buoyant-density membranes were prepared by detergent extraction essentially as described (23). Briefly, osteoblasts were lysed on ice for 30 min in 1 ml of extraction buffer [25 mM HEPES, pH 7.0, 150 mM NaCl, 1% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µg/ml o-phenanthroline, 10 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride, and 50 µg/ml trans-epoxysuccinyl-L-leucinamido(4-guanidino)butane]. Cell lysates were collected by scraping and were passed through a 21-gauge needle 10 times. The lysates were mixed with an equal volume of 80% sucrose in 25 mM HEPES and placed in the bottom of a centrifuge tube. After a continuous layer of sucrose (35–5%) was overlaid on the lysates, the tubes were centrifuged at 100,000 g for 18 h at 4°C in an SW 55 rotor. Fractions (0.5 ml) were collected beginning at the top of the gradient. A 20-µl sample from each fraction was prepared for Western blot analysis as described below.

Western blot analysis. Proteins were prepared in sample buffer [170 mM Tris·HCl, pH 6.8, 3% (wt/vol) SDS, 1.2% (vol/vol) {beta}-mercaptoethanol, 2 M urea, and 3 mM EDTA] and separated by SDS-PAGE (10% gel) before electrotransfer to nitrocellulose filters for immunoblotting. Filters were incubated with indicated primary antibodies followed by either anti-mouse or anti-rabbit horseradish peroxidase secondary antibody. Proteins were detected using enhanced chemiluminescence substrate (ECL, Amersham). Densitometric quantification of immunoblots using ImageQuant software (Molecular Dynamics, Sunnyvale, CA) was performed to enable direct comparisons of ECL signals between each group. Protein content of each sample was determined by bicinchoninic acid analysis.

Statistical analysis. At least three independent experiments were conducted for each study. Raw data from individual experiments were collected and pooled according to group (i.e., control, pressure-treated, shear-treated, CD-filipin-pretreated, or CD-filipin-pretreated cells exposed to pressure or shear). Means and standard deviations were calculated for each group and analyzed with unpaired two-tailed Student's t-test using Statgraphics 4.0 software (Statistical Graphics). Differences between control and experimental groups were deemed to be significant at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hydrostatic pressure activates signaling transduction in osteoblasts. Osteoblast responsiveness to fluid mechanical stimulation was measured by analysis of specific signal transduction parameters that describe mechanotransduction events. We observed that total protein tyrosine phosphorylation increased in osteoblasts exposed to sustained hydrostatic pressures of 5 and 10 mmHg compared with cells maintained at ambient pressure. Compared with results obtained from control cells, protein tyrosine phosphorylation increased nearly 3.5-fold in cells subjected to 5 mmHg pressure and 7.3-fold at 10 mmHg for 10 min (Fig. 1). This pressure-induced increase in protein tyrosine phosphorylation appeared to be transient; osteoblasts subjected to 5 mmHg of hydrostatic pressure sustained for 30 min showed no significant difference in phosphotyrosine levels compared with control (Fig. 1). Although a higher magnitude of hydrostatic pressure (10 mmHg) could still induce significant levels of protein tyrosine phosphorylation (3.6-fold at 10 mmHg for 30 min), these values were ~50% lower than those observed at the 10-min observation point (Fig. 1). These data demonstrate that osteoblasts are sensitive to both the magnitude and duration of externally applied sustained hydrostatic pressures.



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Fig. 1. Enhanced protein tyrosine phosphorylation in osteoblasts in response to hydrostatic pressure. Osteoblasts were exposed to either 5 or 10 mmHg of hydrostatic pressure sustained for 10 or 30 min. Whole cell lysates were resolved, transferred, and immunoblotted with phosphotyrosine antibody 4G10. A: Western blot shows that the profile of tyrosine-phosphorylated (pY) proteins increased after 10- or 30-min exposure to hydrostatic pressure. B: total pY detected after exposure to each pressure magnitude for 10 and 30 min was densitometrically quantified. These results indicate that pressure-induced protein tyrosine phosphorylation occurred in a transient manner. Extent of protein tyrosine phosphorylation was also dependent on the magnitude of the sustained hydrostatic pressure applied to the cells. For these experiments, {beta}-actin showed no change in expression and thus served as a load control. Data are reported as percent fold increases over cells maintained at ambient pressure and are expressed as means ± SD from three independent experiments; *P < 0.05.

 

A convergence point for many of the second-messenger signaling pathways is the mitogen-activated protein kinases (MAPKs). Activation of both 42- and 44-kDa MAPKs [extra-cellular signal-regulated kinase (ERK)1/2] occurs in endothelial cells in response to increased fluid shear stress (12, 46). Similar to our observations of pressure-induced protein tyrosine phosphorylation, 10 min of sustained hydrostatic pressure enhanced ERK1/2 activity 3.2- and 8.3-fold under sustained pressures of 5 and 10 mmHg, respectively (Fig. 2). After a 30-min exposure to 5 mmHg of pressure, ERK1/2 phosphorylation measured only 1.6-fold over control, whereas a pressure of 10 mmHg enhanced induced ERK1/2 activation by fourfold (Fig. 2). Hence activation of downstream ERK1/2 MAPKs in osteoblasts also appears to be sensitive to both time and pressure magnitude.



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Fig. 2. Hydrostatic pressure-induced extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) activation within osteoblasts. Osteoblasts were exposed to sustained hydrostatic pressures of either 5 or 10 mmHg for 10 and 30 min. Whole cell lysates were resolved, transferred, and immunoblotted with a phosphospecific antibody for ERK1/2. A: Western blot shows that sustained hydrostatic pressures rapidly activated ERK1/2. B: phosphorylated ERK1/2 (pERK1/2) detected after each time point of applied pressure was densitometrically quantified. Similar to the results obtained for protein tyrosine phosphorylation, pressure-induced activation of ERK1/2 MAPK occurred in a transient manner. Extent of activation was dependent on the magnitude of the hydrostatic pressure applied to the cells. ERK2 served as a load control for these experiments. Data are reported as percent fold increases over cells maintained at ambient pressure and are expressed as means ± SD from three independent experiments; *P < 0.05.

 

The early-response gene c-fos is believed to play an important role in bone cell physiology through regulation of transcription factors such as activator protein-1, which can engage genes for collegen type I, osteocalcin, osteopontin, and others (31, 45). Here we show that hydrostatic pressure enhanced c-fos expression in both a time- and pressure-dependent manner. Expression of c-fos increased 2.2-fold in osteoblasts exposed to 5 mmHg of pressure for 10 min (Fig. 3). By 30 min, expression of c-fos was 3.5-fold greater than that observed in control cells (Fig. 3). Under sustained hydrostatic pressure of 10 mmHg, c-fos expression increased 3.8-fold after 10 min, and by 30 min, expression was enhanced by nearly fivefold compared with cells exposed to ambient pressure (Fig. 3).



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Fig. 3. Hydrostatic pressure enhances expression of the early response gene c-fos within osteoblasts. Osteoblasts were exposed to sustained hydrostatic pressures of either 5 or 10 mmHg for 10 and 30 min. Whole cell lysates were resolved, transferred, and immunoblotted with c-fos antibody. A: Western blot shows the c-fos expression induced by exposure of osteoblasts to sustained hydrostatic pressures for either 10 or 30 min. B: total expression detected at each time point and for each magnitude of pressure was densitometrically quantified. Expression of c-fos was sensitive to the magnitude of pressure applied to osteoblasts. Each level of sustained hydrostatic pressure tested enhanced c-fos expression as early as 10 min and as late as 30 min. {beta}-Actin showed no change in expression and thus served as a load control. Data are reported as percent fold increases over cells maintained at ambient pressure and are expressed as means ± SD from three independent experiments; *P < 0.05.

 

CD and filipin alter hydrostatic pressure-induced mechanotransduction. Because cholesterol is essential for structural maintenance of lipid rafts and caveolar-signaling complexes, cholesterol-binding agents provide a useful pharmacological tool for evaluating cholesterol-rich plasma membrane microdomain function. In this study, we pretreated osteoblasts with the cholesterol-sequestering compound CD before exposing the cells to hydrostatic pressure. Incubation of osteoblasts with 1 mM CD for 30 min did not alter cell morphology or integrity as measured by Trypan blue exclusion (data not shown). Figure 4 shows that pressure-induced protein tyrosine phosphorylation, ERK1/2 activation, and enhanced c-fos expression were significantly reduced and approached nontreated, unstimulated baseline values measured in control cells after depletion of plasma membrane cholesterol. To further investigate the role of cholesterol in spatiotemporal mechanosignaling, CD-pretreated osteoblasts were incubated with cholesterol-loaded CD to replenish the plasma membrane cholesterol content. Figure 5 illustrates that cholesterol repletion restored pressure-induced signaling.



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Fig. 4. Depletion of plasma membrane cholesterol attenuates pressure-induced mechanosignaling in osteoblast cells. Osteoblasts were incubated with either methyl-{beta}-cyclodextrin (CD) for 30 min or filipin for 5 min before exposure to 10 mmHg hydrostatic pressure sustained for 10 min. Whole cell lysates were resolved, transferred, and immunoblotted with respective primary antibodies. Lipid raft/caveolae disassembly attenuated tyrosine phosphorylation, ERK1/2 activation, and c-fos expression in response to hydrostatic pressure. {beta}-Actin and ERK2 served to demonstrate equivalent amount of protein loaded in each lane. Western blot is representative of three independent experiments.

 


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Fig. 5. Reconstitution of cholesterol-enriched rafts/caveolae restores pressure-induced mechanotransduction. A: to restore cholesterol-rich raft domains, osteoblast cell cultures were incubated with a cholesterol-loaded CD for 1 h at 37°C as described in METHODS. After raft reconstitution, osteoblasts were subjected to 10-mmHg hydrostatic pressure sustained for 10 min. Whole cell lysates were resolved, transferred, and immunoblotted with anti-phosphotyrosine antibody 4G10. Allowing lipid rafts/caveolae to reassemble restores the pressure-induced mechanotransduction responses attenuated by acute CD treatment. Western blot is representative of three independent experiments.

 

In a complementary set of experiments, osteoblasts were pretreated with the alternative raft-disrupting agent filipin. Similar to CD, filipin pretreatment significantly reduced mechanosignaling in these cells (see Fig. 4). These data together with the CD data suggest that intact cholesterol-rich plasma membrane compartments (i.e., lipid rafts and caveolae) are important components of the pressure-induced mechanotransduction pathway in osteoblasts.

Pressure-induced mechanosignaling events occur within caveolin-containing membrane microdomains. To begin to assess whether mechanotransduction events occur within caveolar membranes, we used sucrose-gradient centrifugation to separate detergent-resistant rafts, which include caveolae, from osteoblast cell lysates. Consistent with past reports (23, 35), the caveolar marker protein caveolin was primarily detected in a light-buoyant-density fraction collected from the 20% sucrose level of the continuous gradient (Fig. 6). As observed in Fig. 1, pressure induced the tyrosine phosphorylation of a variety of cellular proteins (Fig. 6). Of these phosphoproteins, bands corresponding to ~115, 85, 60–58, and 24 kDa were enriched in the caveolin-enriched lipid raft fraction of the sucrose gradient. In addition, proteins of apparent 180, 120, 70, and 40 kDa were sedimented in higherdensity nonraft cellular compartments. Thus a portion of the proteins that were tyrosine-phosphorylated in response to increased hydrostatic pressure were detected within caveolin-enriched plasma membrane rafts. This demonstrates that upstream signaling events can occur within these microdomains.



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Fig. 6. Pressure-induced tyrosine-phosphorylated proteins reside in caveolin-enriched membranes. Control and pressure-challenged osteoblasts were lysed by detergent extraction and fractionated on a continuous sucrose gradient to yield caveolinenriched membranes as described in METHODS. A: a sample of each fraction was loaded by equivalent volume onto a 10% SDS-PAGE gel. Proteins were then separated and transferred to nitrocelluose membranes. Western blot analyses showed caveolin-1 to be enriched in the low-buoyant-density fractions. The same fractions probed with anti-phosphotyrosine (4G10) showed several proteins tyrosine-phosphorylated in response to hydrostatic pressure localized in the same membrane fraction as caveolin. Experiments shown are representative of at least three independent experiments. B: a protein concentration profile for each fraction of the sucrose gradient collected from control and pressure-treated osteoblast cultures is shown.

 

Shear stress activates mechanosignaling in osteoblasts through cholesterol-rich signaling compartments. Because fluid shear stress is also considered to be an important factor in regulation of osteoblast function in vivo, we examined mechanotransduction responses in osteoblasts subjected to laminar shear stress. Exposure to shear stress (10 dyn/cm2) for 10 min induced a 5.2-fold increase in protein tyrosine phosphorylation over sham-manipulated osteoblasts (Fig. 7). In addition, ERK1/2 activity increased 2.5-fold (Fig. 8) and c-fos expression was enhanced by fourfold (Fig. 9) in response to laminar shear stress. Thus both shear stress and sustained hydrostatic pressure initiate similar mechanotransduction responses that lead to enhanced gene expression in osteoblasts.



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Fig. 7. Shear stress-induced protein tyrosine phosphorylation is dependent on intact lipid raft/caveolae. Osteoblasts were pretreated with either CD (1 mM) or vehicle at 37°C for 30 min, placed in a parallel device, and exposed to laminar shear stress (10 dyn/cm2) for 10 min. Whole cell lysates were resolved, transferred, and immunoblotted with phosphotyrosine antibody 4G10. A: shear stress induced tyrosine phosphorylation of several cellular proteins. CD pretreatment substantially reduced this response. B: densitometric quantification of total protein tyrosine phosphorylation shows a fivefold increase in osteoblasts exposed to shear stress. Lipid raft/caveolae ablation reduced this response by ~50%. {beta}-Actin showed no change in expression and thus served as a load control. Data are reported as percent fold increases over shams and are expressed as means ± SD from three independent experiments. *P < 0.05; #P < 0.05.

 


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Fig. 8. Shear stress-induced ERK1/2 activation is dampened after cholesterol sequestration. Osteoblasts were pretreated with either CD (1 mM) or vehicle at 37°C for 30 min, placed in a parallel device, and exposed to laminar shear stress (10 dyn/cm2) for 10 min. Whole cell lysates were resolved, transferred, and immunoblotted with an antibody that detects activated ERK1/2. A: shear-induced ERK1/2 activation was attenuated by CD pretreatment. B: laminar shear stress enhanced ERK1/2 activity by ~2.5-fold but only by 1.5-fold for osteoblasts pretreated with CD. ERK2 served as a load control. Data are reported as percent fold increases over shams and are expressed as means ± SD from three independent experiments. *P < 0.05; #P < 0.05.

 


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Fig. 9. Shear stress-induced c-fos expression is reduced after CD pretreatment. Osteoblasts were pretreated with either CD (1 mM) or vehicle at 37°C for 30 min, placed in a parallel device, and exposed to laminar shear stress (10 dyn/cm2) for 10 min. Whole cell lysates were resolved, transferred, and immunoblotted with c-fos antibody. A: CD pretreatment significantly reduced shear stress-induced c-fos expression. B: densitometric quantification of c-fos expression shows a fourfold increase in c-fos expression in osteoblasts exposed to shear stress. Lipid raft/caveolae ablation reduced this response by ~35%. {beta}-Actin showed no change in expression and thus served as a load control. Data are reported as percent fold increases over shams and are expressed as means ± SD from three independent experiments. *P < 0.05.

 

To evaluate whether intact cholesterol-rich plasma membrane microdomains participate in the shear-induced mechanotransduction response, osteoblast membrane cholesterol was extracted (see Lipid raft and caveolae manipulation with cholesterol-interacting agents). Similar to our observations for sustained hydrostatic pressure-induced mechanotransduction, lipid raft and caveolae ablation significantly reduced the capacity of the osteoblast to propagate a shear-induced mechanotransduction signaling cascade. Compared with nontreated cells, shear-induced protein tyrosine phosphorylation (see Fig. 7), ERK1/2 activation (see Fig. 8), and enhanced c-fos expression (see Fig. 9) were stimulated to approximately half of the value in cells treated with CD. Taken together, these data suggest that osteoblasts use cholesterol-rich lipid rafts and/or caveolae signaling compartments for activation of hydrostatic pressure- and shear stress-responsive mechanotransduction pathways.


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We first examined responsiveness of osteoblasts to changes in the fluid-mechanical environment by using in vitro systems designed to mimic biomechanical forces generated during bone loading. Previous in vitro studies showed that hydrostatic pressure stimulates production of prostaglandins and other signaling molecules, activity of alkaline phosphatase, and expression of c-fos and the osteoblastic marker genes collagen and osteopontin (9, 14, 32). Here we show that exposure of osteoblasts to sustained hydrostatic pressure stimulated additional mechanosignaling pathways that included protein tyrosine phosphorylation and ERK1/2 MAPK activation (see Figs. 1 and 2). Consistent with past reports (9), we also observed that osteoblasts subjected to increased magnitudes of pressure responded by enhancing transcriptional expression of c-fos, the early regulatory gene, in a time-dependent manner (see Fig. 3).

Increasing evidence shows that fluid shear stress also plays an important role in regulating osteoblast metabolism (13, 16, 2427, 39). Osteoblasts derived from a variety of bone types produce both NO and PGE2 in response to shear stress (13, 16, 39). Application of shear stress to mouse osteoblastic MC3T3-E1 cells enhanced expression of COX-2 and c-fos (5, 22) through a mechanotransduction pathway that involved IP3-mediated intracellular Ca2+ release (5). In the present study, exposure to laminar shear stress (10 dyn/cm2 for 10 min) activated additional signaling pathways in osteoblasts. Both protein tyrosine phosphorylation (see Fig. 7) and activation of ERK1/2 MAPK were enhanced by shear stress (see Fig. 8). In agreement with previous studies (5, 22), we observed that fluid shear stress induced a mechanotransduction cascade that led to enhanced transcription of c-fos (see Fig. 9). Thus osteoblasts are capable of sensing compressive and shear forces, which are subsequently converted into biochemical signaling through activation of second-messenger signaling molecules.

We observed that both hydrostatic pressure and laminar shear stress activated comparable mechanotransduction pathways including a similar pattern of protein tyrosine phosphorylation, ERK1/2 activation, and enhanced c-fos expression. However, osteoblasts showed greater mechanosensitivity to hydrostatic pressure than shear stress. After 10 min, 10 mmHg of hydrostatic pressure increased protein tyrosine phosphorylation by 7.3-fold, whereas 10 min of laminar shear stress applied at 10 dyn/cm2 stimulated a 5.2-fold increase in phosphorylation. Likewise, hydrostatic pressure induced an eight-fold activation of ERK1/2 after 10 min, whereas osteoblasts subjected to shear stress for the same duration of time enhanced ERK1/2 activity by 2.5-fold. Although these observations indicate that osteoblasts are more responsive to changes in pressure, whether changes in shear magnitude would alter the degree of the mechanotransduction response in these cells remains to be tested. Because osteoblasts are subjected to both pressure and shear forces in vivo, direct comparisons between the effects that these forces exert on osteoblast function would provide greater insight into the role that fluid mechanical forces play in bone homeostasis.

Although our present observations in conjunction with previous studies investigating the mechanotransduction process in osteoblasts and other cell types serve to elucidate the second-messenger signaling molecules that propagate force transmission within the cell, the initial force-sensing element(s) or mechanoreceptor(s) remains elusive. To date, the most intensively studied possible mechanosensing structures has been the focal adhesion regions of the cell membrane (for overview, see Ref. 6). Focal adhesions play a role in the adaptations of cultured endothelial cells. These sites undergo remodeling when shear stress is applied to the luminal surface of cultured endothelial cells and realign in the direction of flow (8). Cytoskeletal elements similarly rearrange and form stress fibers parallel to the direction of flow, which suggests an association with focal adhesions (8). In addition, several cytosolic proteins localized to focal adhesions, particularly focal adhesion kinase (34) and paxillin (47), undergo phosphorylation and initiate signal transduction events in response to flow. In osteoblasts, shear-induced recruitment of {alpha}-actinin and {beta}1-integrin to focal adhesion sites that correlated with enhanced expression of COX-2 and c-fos (22) suggested a link between focal adhesion signaling events and gene expression. Although focal adhesion complexes can clearly participate in the cellular responses to flow, they do not give a complete account of the mechanism(s) involved in the mechanotransduction process within either endothelial cells or osteoblasts.

It is becoming increasingly appreciated that the plasma membrane contains organized regions or microdomains that have a unique lipid and protein composition. These domains, known as lipid rafts, are assembled primarily of glycerolphospholipids and enriched with cholesterol and sphingolipids (23). Caveolae are considered specialized rafts due to the association of caveolin proteins with raft lipids (40, 44). These structures are particularly prominent in adipocytes and endothelial, smooth muscle, and epithelial cells and have recently been described in osteoblasts (15, 42). Both rafts and caveolae are purported to participate in diverse cellular functions including cholesterol transport (7, 41), endocytosis (36), and potocytosis (1). In addition, several laboratories have reported that many receptors and signaling molecules are concentrated within these cholesterol-enriched membrane compartments (4, 38, 40). This latter observation has led to the hypothesis that these membrane regions serve as platforms for organizing and integrating signal transduction processes within the cell. Indeed, this concept has now been demonstrated in a wide variety of cell types including osteoblasts (43). Solomon et al. (43) showed that platelet-derived growth-factor receptors, nonreceptor tyrosine kinases, tyrosine adapter proteins, G proteins, and NO synthases were clustered as a signaling complex in caveolin-enriched membrane fractions derived from human and murine osteoblasts. More importantly, ligand-stimulated activation of platelet-derived growth-factor receptor-induced phosphorylation and dynamic regulation of MAPK effectors located within these membrane fractions (43) suggest that caveolin-enriched membrane domains are important sites for signaling transduction in osteoblasts.

Because many of the signaling molecules that reside in lipid rafts and caveolae have been implicated in the transduction of fluid mechanical forces into chemical signals, we hypothesized that cholesterol-rich plasma membrane microdomains, particularly caveolae, serve as logical sites for initiation of a mechanotransduction signaling cascade. We first tested this concept in an in situ lung perfusion model (30). We reported that increasing flow and pressure in rat lungs in situ stimulated protein tyrosine phosphorylation at the luminal endothelial plasma membrane specifically within caveolae (30). Enhancing flow through the rat lung vasculature also activated endothelial NO synthase located in caveolae (28) and a signaling cascade that involved Ras-Raf-ERK1/2 (30). In addition, we and others recently observed that flow preconditioning of cultured endothelial cells enhanced the density of cell surface caveolae and recruited them into mechanotransduction pathways (3, 29). From these studies, we concluded that caveolae serve as mechanotransduction sites at the endothelial cell surface. Because osteoblasts are subjected to similar biomechanical forces (i.e., pressure and shear stress) and appear to respond to these forces using similar signaling mechanisms as endothelial cells, we tested whether cholesterol-rich plasma membrane microdomains play a role in the mechanotransduction process in this cell type.

Cholesterol binding agents such as cyclodextrins and filipin sequester cholesterol and cause disassembly of cholesterol-rich rafts and caveolae (11, 21, 33). Thus these compounds provide useful tools for study of lipid raft and caveolae functions. Consistent with previous reports that demonstrated that CD acts as a nonintrusive agent for reduction of plasma membrane cholesterol (11), osteoblasts incubated with CD showed no change in cell morphology, viability, or baseline levels of the signaling parameters monitored in this study (Figs. 4 and 7, 8, 9, as well as data not shown). Here we provide evidence that disruption of cholesterol-rich plasma membrane compartments significantly reduce both pressure and shear-induced mechanotransduction events in osteoblasts (Figs. 4 and 7, 8, 9). More importantly, mechanotransduction responses could be restored in osteoblasts where rafts were reconstituted (see Fig. 5). These results are in support of our past studies, which showed that abrogation of lipid rafts and caveolar architecture with cholesterol-sequestering compounds served to prevent mechanotransduction in rat lungs (30). Furthermore, Park et al. (21) reported that cholesterol depletion with CD also prevented shear-induced activation of MAPK in cultured endothelial cells. Taken together, these data support the concept that lipid rafts and caveolae function as important mechanotransduction sites in both endothelial cells and osteoblasts.

To begin to evaluate whether the observed mechanotransduction responses occurred directly in caveolar raft domains, we isolated caveolin-enriched membranes from control and pressure-treated osteoblasts and probed for mechanotransduction events. Here we show that several proteins phosphorylated on tyrosine in response to enhanced pressure were localized in caveolin-containing rafts (see Fig. 6) including an apparent 24-kDa protein. Although the identities of these proteins remain unknown, caveolin-1 is rapidly phosphorylated on Tyr14 in response to shear stress in endothelial cells (29). Thus it is tempting to speculate that the apparent 24-kDa protein is tyrosine-phosphorylated caveolin-1. The presence of mechano-sensitive proteins, potentially including caveolin-1, demonstrates that at least some mechanotransducing elements reside within these microdomains. Interestingly, we observed the caveolin-1 signal trending toward the lipid raft fractions of the sucrose gradient after pressure exposure. Whether increased caveolin levels in the lipid raft fraction represent a recruitment of caveolae into mechanotransduction pathways similar to that observed for endothelial cells subjected to shear stress (3, 29) remains to be examined.

We also observed a substantial number of phosphoproteins that were not associated within caveolin-enriched rafts (see Fig. 6). The ability of fluid mechanical forces to enhance tyrosine phosphorylation of these proteins, however, was significantly dampened by treatment with both cyclodextrin and filipin. The most obvious interpretation of this finding is that these proteins are localized to noncaveolar lipid raft domains that are mechanosensitive. Another possibility is that these proteins are part of the downstream mechanotransduction process and reside in cellular compartments other than caveolin-containing membranes. In this senario, fluid mechanical forces activate signaling molecules targeted to caveolin-enriched rafts such as Src-like kinases, which then induce phosphorylation of proteins that are not necessarily localized to raft domains. Therefore, ablation of the raft mechanosignaling domain with cholesterol-sequestering compounds may not allow for the subsequent activation of these proteins. Yet another alternative is the association of these phosphoproteins with other mechanosensitive sites within the cell such as focal contacts. Emerging evidence (10, 49, 50) suggests that caveolae and integrin sites possess the ability to cross-talk. In support of this concept, we find that {beta}1-integrin-mediated mechanotransduction is regulated by plasma membrane cholesterol and caveolin-1 in endothelial cells (our unpublished observations).

In summary, hydrostatic pressure and laminar shear stress applied to osteoblast cell cultures stimulate a mechanotransduction cascade that is partially dependent on the integrity of cholesterol-enriched plasma membrane compartments. These conclusions are supported by the observation that two independent and well-characterized pharmacological agents for disrupting cholesterol-rich domains, CD and filipin, blunt fluid mechanical force-induced signal transduction in osteoblasts. Furthermore, osteoblasts regain their mechanotransducing properties after the restoration of plasma membrane rafts. The localization of at least part of the observed mechanotransduction event to caveolin-enriched lipid rafts supports our previous findings that these domains can serve as mechanotransduction sites. Because a fair degree of mechanostimulated molecules appeared to be unassociated with rafts and caveolae but sensitive to cholesterol-sequestering compounds, we do not rule out that other cellular compartments or regions, perhaps influenced by cholesterol-enriched membrane domains, play an additional role in the mechanotransduction process. Our data support the general concept that proper organization of the lipid milieu within plasma membranes allows the cell to compartmentalize lipids and protein-signaling mediators that provide a more efficient means to respond to fluid-mechanical stimulation. Additional studies are necessary to determine whether raft- and caveolae-mediated mechanotransduction events require these structures as a whole or are regulated through a single molecule or multiple effectors located in these microdomains such as G proteins, endothelial NO synthase, and calcium transport proteins. Such studies would be relevant toward understanding the mechanisms by which osteoblasts respond to mechanical forces and thereby influence bone tissue adaptation to the physical environment.


    ACKNOWLEDGMENTS
 
We thank Sheila Dela Cruz (Dept. of Biomedical Engineering, Rensselaer Polytechnic Institute) for instruction and assistance in using the sustained hydrostatic pressure chamber and Tin N. Oo for excellent technical support.

GRANTS

This work was supported by American Heart Association, New York Affiliate Grant 0030300T (to V. Rizzo).


    FOOTNOTES
 

Address for reprint requests and other correspondence: V. Rizzo, Center for Cardiovascular Science, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208 (E-mail: rizzov{at}mail.amc.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.


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