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J Appl Physiol 97: 135-142, 2004. First published March 5, 2004; doi:10.1152/japplphysiol.01260.2003
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Formation of focal adhesions on fibronectin promotes fluid shear stress induction of COX-2 and PGE2 release in MC3T3-E1 osteoblasts

Suzanne M. Ponik and Fredrick M. Pavalko

Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202

Submitted 24 November 2003 ; accepted in final form 27 February 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Mechanical loading of bone is important for the structural integrity of the skeleton and the maintenance of bone mass. Mechanically loading bone generates fluid shear stress (FSS) across the surface of bone cells resulting in the induction of cyclooxygenase-2 (COX-2) and release of prostaglandins, both of which are necessary for mechanically induced bone formation. However, the mechanisms by which cells transduce FSS-induced signals across the membrane and into the cell remain poorly understood. Focal adhesions, which are specialized sites of attachment between cells and the extracellular matrix, play a role in signal transduction and have been proposed to function as mechanosensors. To directly test whether focal adhesions mediate mechanotransduction in bone cells, we inhibited the formation of focal adhesions by 1) culturing MC3T3-E1 osteoblasts on bovine serum albumin (BSA), which does not contain integrin binding sites or by 2) treating cells cultured on fibronectin with soluble Arg-Gly-Asp-Ser (RGDS) peptide to specifically block integrin-fibronectin interactions. We then subjected the cells to FSS and measured COX-2 induction and PGE2 release. Both COX-2 induction and PGE2 release in response to FSS were significantly decreased when osteoblasts were treated with soluble RGDS peptide compared with controls. However, RGDS peptide treatment did not affect FSS-induced ERK phosphorylation. Interestingly, osteoblasts cultured on BSA to suppress focal adhesion formation secreted fibronectin and increased focal adhesion formation over time, which correlated with the induction of COX-2 in response to FSS. Together, these results suggest that fibronectin-induced formation of focal adhesions promotes FSS-induced PGE2 release and upregulation of COX-2 protein.

integrins; bone; mechanical loading; cyclooxygenase-2; prostaglandin E2; metabolism

BONE IS A DYNAMIC TISSUE THAT models and remodels in response to mechanical loads from the external environment (16, 23, 33, 38). At the cellular level, mechanical loading of bone generates movement of interstitial fluid, creating fluid shear stress across the surface of bone cells (8, 9, 26). In response to fluid shear stress, osteoblasts increase expression of cyclooxygenase-2 (COX-2) and increase release of prostaglandin E2 (PGE2) (2, 21, 30–32, 37), which are required for mechanically induced bone formation (6, 12). However, the mechanisms by which osteoblasts detect fluid shear stress and transduce the signal across the membrane to activate signaling pathways involved in bone metabolism, such as induction of COX-2 and release of PGE2, remain poorly understood.

Focal adhesions are specialized sites of cell-matrix interaction composed of integrins and many focal adhesion-associated cytoplasmic proteins including, but not limited to, vinculin, focal adhesion kinase (FAK), {alpha}-actinin, and actin filaments (3). Because of the cellular location of focal adhesions, it has been suggested that focal adhesions function as mechanoreceptors. Specifically, binding of integrins to the extracellular matrix may facilitate the transfer of mechanical signals from the external environment across the membrane to activate intracellular signaling cascades (1, 14, 18, 36). Experimental evidence supporting a role for focal adhesions as mechanoreceptors includes the observation that fluid shear stress activates intracellular focal adhesion components. For example, fluid shear stress induces integrin-Shc association, depolarization of the membrane, and increased phosphorylation of FAK. Each of these processes can be inhibited by integrin function-blocking antibodies or soluble Arg-Gly-Asp-Ser (RGDS) peptides that inhibit integrin binding to the RGDS sequence within many matrix proteins (19, 22, 24, 34). In addition, fluid shear stress can also cause morphological changes to focal adhesions, including increased size and number of focal adhesions and increased recruitment of {beta}1-integrin to focal adhesions (7, 30). Although integrin activation clearly occurs in response to fluid shear stress, the induction of cyclooxygenase-2 (COX-2) and release of prostaglandins in response to fluid shear stress, which are necessary for mechanically induced bone formation, have not been directly linked to integrins or focal adhesion formation.

In this study, we investigated the role of focal adhesions as potential mechanosensors through which fluid shear stress-induced mechanical signals may be transduced across the osteoblast cell membrane to activate PGE2 release and induce COX-2 protein expression. We found that inhibiting focal adhesion formation by culturing osteoblasts on bovine serum albumin (BSA), a substrate that does not contain integrin binding sites, significantly reduced PGE2 release in response to fluid shear stress. When soluble RGDS peptide was used to inhibit integrin binding and limit focal adhesion formation on fibronectin, fluid shear stress-induced PGE2 release as well as upregulation of COX-2 protein were significantly inhibited. However, fluid shear stress-induced ERK phosphorylation was unaltered when focal adhesion formation was inhibited with the use of soluble RGDS peptide. These results suggest that limiting focal adhesion formation inhibits fluid shear stress-induced PGE2 release and upregulation of COX-2 protein but not fluid shear stress-induced ERK phosphorylation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Cell culture conditions.   MC3T3-E1 osteoblasts were grown in {alpha}-MEM containing 10% fetal calf serum with 1% penicillin and streptomycin. To modulate focal adhesion formation, glass slides were coated with a solution of BSA (10 µg/cm2) or bovine plasma fibronectin (0.7 µg/cm2) (Sigma-Aldrich) and allowed to dry completely. After the slides were rinsed with PBS, MC3T3 osteoblasts were passaged onto the BSA- or fibronectin-coated slides and allowed to adhere for 2 h in {alpha}-MEM containing 0% fetal calf serum and 1% penicillin and streptomycin. Serum was excluded to eliminate the effect of matrix proteins contained in serum and the length of time in culture was kept short to minimize the influence of cell-secreted matrix proteins. RGDS or Arg-Gly-Glu-Ser (RGES) peptide (Sigma-Aldrich) treatment included incubating suspended MC3T3 osteoblasts for 1 h while they rotated at 4° in {alpha}-MEM containing 0% fetal calf serum and 500 µg/ml RGDS or RGES peptide or an equal volume of vehicle (PBS). After the 1-h treatment, osteoblasts were allowed to adhere to fibronectin-coated slides in the continued presence of RGDS or RGES peptide for 2 h before static or fluid shear stress conditions in the continued presence of vehicle or peptide.

Fluid flow conditions.   Cells were subjected to laminar fluid shear stress (10 dyn/cm2) by using parallel-plate flow chambers and the flow-loop system designed by Frangos et al. (13) and marketed by Cytodyne (San Diego, CA). Fluid shear stress experiments were performed at 37°C with 25 ml of {alpha}-MEM subjected to a stream of 5% CO2. Static controls were incubated in the same volume of media at 37°C with 5% CO2. Cells cultured for 4 days in {alpha}-MEM containing 10% serum were subjected to a time course of fluid shear stress in {alpha}-MEM containing 1% serum. Serum contains 3–6 g protein/100 ml (3–6% protein). Therefore, when we subject cells to fluid shear stress in the presence of 1% serum, we are using a total protein concentration of 0.03–0.06% protein. To verify that the absence of protein did not have any adverse effects on the cells, we also cultured cells for 2 h on fibronectin-coated slides in {alpha}-MEM containing 0% serum or 0.1% BSA and then subjected the cells to a time course of fluid shear stress in {alpha}-MEM containing 0% serum or 0.1% BSA, respectively. We chose 0.1% BSA because this is close to the concentration of protein in media containing 1% serum. After verification that there was no difference in the level of COX-2 expression in the presence or absence of protein in the media (see Fig. 1), subsequent experiments in this study were performed in the absence of serum or BSA and included 2 h of adhesion followed by 5 h of fluid shear stress or static culture conditions.



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  		Fig. 1. Western blot analysis of cyclooxygenase-2 (COX-2) induction in response to fluid shear stress under modified culture conditions. A: cells were cultured in media containing 10% fetal calf serum for 4 days on glass slides before a time course of fluid shear stress in media containing 1% serum. Under these conditions, COX-2 protein levels significantly increased after 1 h of fluid shear stress (P < 0.05, 0 min vs. 1 h and 1.5 h, by Student's t-test). B: cells were cultured for 2 h on glass slides coated with fibronectin (FN) before a time course of fluid shear stress in the absence of serum. A significant increase in COX-2 protein was detected under these conditions after 5 h of fluid shear stress (P < 0.05, 0 min vs. 5 h, by Student's t-test). C: cells were cultured for 2 h on glass slides coated with FN before a time course of fluid shear stress in serum-free media containing a nonmatrix protein [0.1% bovine serum albumin (BSA)]. A significant increase in COX-2 protein was detected under these conditions after 5 h of fluid shear stress (P < 0.05, 0 min vs. 5 h, by Student's t-test). Densitometric analysis of 3 experiments was performed for all time points and culture conditions.

 
Immunofluorescence microscopy.   For fluorescence microscopy, cells were fixed in 4% paraformaldehyde for 15 min, permeabilized in 0.2% Triton, and processed with the appropriate antibody. We record images with a RT Color Spotdigital camera (Diagnostic Instruments) attached to an Optiphot-2 Nikon epifluorescent microscope that used a x60 Nikon planapo objective.

Immunoblot.   For immunoblot analysis, cells subjected to either static culture or fluid shear stress were harvested in SDS sample buffer. Protein concentrations were determined by using the amido black method (35). Equal cellular protein (40 µg) was loaded onto SDS-PAGE gels for separation and transferred to nitrocellulose for immunoblotting. The antibody signal was detected and analyzed with a chemiluminescence imager (Fuji, Edison, NJ) after reaction in a luminescence solution described in Norvell et al. (27). The experiments were carried out in at least triplicate.

Antibodies.   For immunofluorescence analysis, we used vinculin VIN 11-5 (Sigma-Aldrich), anti-{alpha}5-integrin 1928 (Chemicon), and anti-fibronectin (Transduction Laboratories). For immunoblot analysis we used, polyclonal COX-2 antibody (Cayman Chemical), monoclonal anti-actin AC-40 (Sigma-Aldrich), anti-ERK1/2 and anti-phospho-ERK1/2 (Santa Cruz). The appropriate secondary antibodies were purchased from Jackson Immunoresearch.

Prostaglandin measurement.   Prostaglandin release was measured after 5 h of fluid flow or static culture by incubating the cells in 1 ml of fresh {alpha}-MEM with 0% fetal calf serum at 37°C and 5% CO2 for 30 min. The media samples were centrifuged at 14,000 g for 1 min to pellet any particulates. The supernatant was transferred to a new tube, and the level of PGE2 present in sample was determined with enzyme immunoassay kits from Amersham Pharmacia Biotech.

Statistical analysis.   Statistical analysis was performed with the statistical package Statview (version 5.0.1). Differences between static and flow and between flow-fibronectin and flow-BSA were tested by using Student's t-test for unpaired variants. Differences among group means were tested for significance by ANOVA, followed by Fisher's protected least significant difference test for all pairwise comparisons. (P values < 0.05 were considered significant).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Induction of COX-2 protein in response to fluid shear stress is delayed under conditions used to modify focal adhesions.   To modulate the formation of focal adhesions in MC3T3-E1 osteoblasts, cell culture conditions were carefully defined. First, serum was excluded from the media to eliminate the confounding effect that serum matrix proteins would have if these proteins were present and allowed to adsorb to the substrate. Second, the length of time in culture was kept short to minimize the potential impact of matrix proteins secreted by osteoblasts and incorporated into the cellular microenvironment on cell-substrate interactions. We first compared COX-2 protein levels during a time course of fluid shear stress under normal-serum and serum-free conditions. We observed a dramatic difference in the length of time required for fluid shear stress to elicit a significant increase in COX-2 protein expression between these two conditions (Fig. 1). Cells cultured on glass slides under conditions that allowed the cells to secrete their own native matrix (4 days in the presence of 10% serum) upregulated COX-2 protein within 30–60 min after the onset of fluid shear stress (10 dyn/cm2) in media containing 1% serum (Fig. 1A). However, cells cultured for only 2 h in the absence of serum on slides coated with fibronectin required 3–5 h of fluid shear stress to elicit an increase in COX-2 protein (Fig. 1B).

To determine whether the absence of protein in the media may have adverse effects on the cells response to fluid shear stress (17), we cultured osteoblasts for 2 h on fibronectin-coated slides and completed a time course of fluid shear stress in the presence of media containing 0.1% BSA. Similar to our results shown in Fig. 1B in which no protein was present in the media, we found that cells cultured on fibronectin for 2 h in the presence of 0.1% BSA also required 3–5 h of fluid shear stress to elicit an increase in COX-2 protein (Fig. 1C). Therefore, to assess the role of focal adhesions as mechanosensors, experiments in this study were performed in the absence of serum and included 2 h of adhesion followed by 5 h of fluid shear stress or static culture conditions.

MC3T3-E1 osteoblasts form robust focal adhesions when cultured on fibronectin but not when cultured on BSA or when treated with soluble RGDS peptide.   To determine the role of focal adhesions in PGE2 release and induction of COX-2 protein in response to fluid shear stress, we used two methods to limit the formation of focal adhesions. MC3T3-E1 osteoblasts were cultured on glass slides coated with BSA or fibronectin in the absence of serum. BSA does not contain integrin binding sites, and therefore this substrate is used to limit focal adhesion formation. Conversely, focal adhesion formation was enhanced by culturing cells on glass slides coated with fibronectin, which contains multiple binding sites for integrins (29). Immunofluorescence analysis using an antibody to detect vinculin positive focal adhesions showed cells were well spread and formed robust focal adhesions after 2 h of adhesion on fibronectin (Fig. 2a). In contrast, cells cultured on BSA adhered and partially spread compared with cells on fibronectin but formed only a few focal adhesions (Fig. 2b).



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  		Fig. 2. Focal adhesion formation under modified culture conditions. Osteoblasts were allowed to adhere to BSA- or FN-coated slides for 2 h or treated with vehicle (PBS), Arg-Gly-Asp-Ser (RGDS), or Arg-Gly-Glu-Ser (RGES) peptide for 1 h in suspension and then allowed to adhere to FN-coated slides for 2 h. Cells were then fixed in 4% paraformaldehyde and permeabilization in 0.2% Triton, and focal adhesions were visualized by using an anti-vinculin antibody. After 2 h, cells cultured on FN formed robust vinculin positive focal adhesions (a). Osteoblasts cultured on BSA failed to form mature focal adhesions and contained only a few small vinculin-positive sites located at the cell periphery (b). Prominent vinculin-positive focal adhesions formed in vehicle- and RGES peptide (500 µg/ml)-treated controls (c and d, respectively). RGDS peptide (500 µg/ml)-treated cells failed to form mature focal adhesion and contained only a few small vinculin-positive sites after 2 h of adhesion (e).

 
Next, to more specifically inhibit focal adhesion formation on a fibronectin matrix, we treated osteoblasts with soluble RGDS peptide. RGDS is the primary integrin binding sequence within fibronectin and therefore treatment with soluble RGDS peptide competes with the RGDS sequence in fibronectin for integrin binding (29). A range of soluble RGDS peptide concentrations were tested, and a concentration of 500 µg/ml RGDS peptide, which did not cause cellular cytotoxicity (data not shown), was chosen for experiments. Visualization of focal adhesions by immunofluorescence microscopy showed treatment with soluble RGDS peptide inhibited the formation of focal adhesions on fibronectin after 2 h of adhesion (Fig. 2e). RGES peptide, which does not bind integrins, was used at the same concentration (500 µg/ml) as a negative control in these experiments. After 2 h in culture osteoblasts treated with vehicle (PBS) (Fig. 2c) or RGES control peptide (Fig. 2d) formed robust focal adhesions.

Fluid shear stress-induced PGE2 release is inhibited by limiting focal adhesion formation.   After 2 h of adhesion we subjected cells to 5 h of fluid shear stress or static culture conditions to determine the role of focal adhesions in fluid shear stress-induced PGE2 release. We found that fluid shear stress-induced PGE2 release was significantly reduced by 47% in cells cultured on BSA compared with cells cultured on fibronectin (Fig. 3A). Treatment of the MC3T3-E1 osteoblasts with soluble RGDS peptide to inhibit the formation of focal adhesions on fibronectin reduced fluid shear stress-induced PGE2 release even more dramatically, by 77 and 78% compared with vehicle-treated controls and RGES peptide controls, respectively (Fig. 3B). Compared with vehicle-treated controls, fluid shear stress-induced PGE2 release was unaltered by treatment with the RGES control peptide (Fig. 3B). These results suggest that the presence of focal adhesions promotes the fluid shear stress-induced release of PGE2.



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  		Fig. 3. Fluid shear stress-induced prostaglandin E2 (PGE2) release is inhibited by limiting focal adhesions. Values are means ± SE. A: in response to 5 h of fluid shear stress, PGE2 release was significantly increased compared with their static counterparts when cultured on BSA or FN. *P < 0.05, Flow vs. Static (by Student's t-test). Fluid shear stress-induced PGE2 release from cells cultured on BSA was decreased by 47% compared with cells cultured on FN. #P < 0.05, Flow-BSA vs. Flow-FN (by Fisher's protected least significant difference test). B: fluid shear stress-induced a significant increase in PGE2 release from FN, FN + RGDS, and FN + RGES cells compared with their static counterparts. *P < 0.05, Flow vs. Static (by Student's t-test). Treatment with RGDS peptide significantly reduced the fluid shear stress-induced release of PGE2 by 77 and 78% compared with vehicle and RGES control cells, respectively. #P < 0.05, Flow-FN + RGDS vs. Flow-FN and Flow-FN + RGES (by Fisher's protected least significant difference test).

 
Fluid shear stress-induced COX-2 upregulation is inhibited by limiting focal adhesion formation on fibronectin with RGDS peptide but not by culturing cells on BSA.   Next, we investigated the role of focal adhesions as mechanosensors in the induction of COX-2 protein in response to fluid shear stress. Surprisingly, when the formation of focal adhesions was inhibited by culturing cells on BSA, 5 h of fluid shear stress increased COX-2 protein levels by 3.8-fold, which was not different from controls (Fig. 4A). Densitometric analysis of the Western blots (n = 6) revealed an average fluid shear stress-induced increase in COX-2 protein of 3.9- and 3.8-fold when cells were cultured on fibronectin and BSA, respectively (Fig. 4C). However, when soluble RGDS peptide was used to inhibit focal adhesion formation on fibronectin, we observed a significant reduction in the fluid shear stress-induced increase in COX-2 compared with both vehicle (PBS)-treated controls and RGES peptide controls (Fig. 4B). The average fold induction of COX-2 measured by densitometry in vehicle-, RGES-, or RGDS-treated cells (n = 4 for each treatment) was 3.9, 3.2, and 2.1 respectively (Fig. 4C).



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  		Fig. 4. Fluid shear stress induction of COX-2 is inhibited by soluble RGDS peptide treatment but not by culturing cells on BSA. A: COX-2 induction significantly increased in response to 5 h of fluid shear stress in cells cultured on both FN and BSA compared with their static counterparts [P < 0.05, Static (S) vs. Flow (F), by Student's t-test]. No difference was detected in fluid shear stress-induced COX-2 protein levels in cells cultured on BSA compared with cells cultured on FN. Equal cellular protein (40 µg) was loaded, and actin was shown as a loading control (n = 6 experiments for densitometry). B: fluid shear stress significantly increased COX-2 protein levels under all treatment conditions compared with their static counterparts (P < 0.05, Flow vs. Static, by Student's t-test). Fluid shear stress-induced COX-2 protein levels in RGES-treated cells were not significantly altered compared with vehicle controls. However, treatment with RGDS peptide significantly decreased fluid shear stress-induced COX-2 protein levels by 46 and 36% compared with vehicle and RGES controls, respectively (P < 0.05, Flow-FN + RGDS vs. Flow-FN and Flow-FN + RGES, by Fisher's protected least significant difference test). Equal cellular protein (40 µg) was loaded and actin was shown as a loading control (n = 5 experiments for densitometry). C: induction of COX-2 protein graphed as the fold increase of flow over static for each culture condition. Values are means ± SE. The average induction of COX-2 was 3.8-, 3.9-, 3.2-, and 2.1-fold in cells cultured on BSA, FN, FN + RGES, and FN + RGDS, respectively. *P < 0.05, FN + RGDS vs. BSA, FN, and FN + RGES (by Fisher's protected least significant difference test).

 
Osteoblasts cultured on BSA secrete fibronectin and form focal adhesions after 5 h of static culture or fluid shear stress.   On the basis of our COX-2 protein expression data, we further examined why cells plated on BSA were able to respond to fluid shear stress, whereas cells treated with soluble RGDS peptide showed a reduced COX-2 response to fluid shear stress. Immunofluorescence analysis of vinculin-positive focal adhesions revealed that in cells cultured on fibronectin neither increasing the amount of time in static culture nor subjecting the cells to 5 h of fluid shear stress altered the formation of focal adhesions (Fig. 5, a–c). In contrast, cells cultured on BSA showed a dramatic increase in vinculin positive focal adhesions after 2 h of adhesion plus 5 h of static culture or fluid shear stress (Fig. 5, e and f) compared with cells allowed to adhere for just 2 h on BSA (Fig. 5d). Thus the formation of focal adhesions in cells cultured on BSA occurred in both static and flow conditions, indicating that the length of time in culture rather than treatment with fluid shear stress induces the formation of focal adhesions.



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  		Fig. 5. MC3T3 osteoblasts grown on BSA secrete FN and form focal adhesions after extended time in culture. Osteoblasts allowed to adhere for 2 h or 2 h plus 5 h of static or fluid shear conditions were fixed in 4% paraformaldehyde and permeabilization in 0.2% Triton. Focal adhesions were visualized by using an anti-vinculin antibody. Osteoblasts cultured on fibronectin formed robust focal adhesion after 2 h of adhesion (a), and the focal adhesions remained prominent after 5 h of static culture or fluid shear stress (b and c). Osteoblasts cultured on BSA had very few mature focal adhesions after 2 h in culture (d) but formed more prominent vinculin-positive focal adhesions after 5 h of static or fluid shear stress conditions (e and f). g–i: Cells were cultured on BSA, and FN secretion was visualized by using an antibody recognizing mouse FN. After 2 h in culture, FN was undetectable in cells cultured on BSA (g). The fluorescence intensity of fibronectin increased after 5 h of static culture or fluid shear stress (h and i).

 
These results led us to investigate whether osteoblasts cultured on BSA might secrete matrix proteins, such as fibronectin, that contain binding sites for focal adhesions. Cells cultured on BSA were fixed after 2 h of adhesion or 2 h of adhesion plus 5 h of static culture or fluid shear stress, and fibronectin secretion was visualized by using an anti-fibronectin antibody. Immunofluorescence analysis showed that MC3T3-E1 osteoblasts were negative for secreted fibronectin after 2 h in culture (Fig. 5g). However, cells cultured on BSA for 2 h and then subjected to 5 h of static culture or fluid shear stress were positive for secreted fibronectin (Fig. 5, h and i), suggesting that cells cultured on BSA may form focal adhesions over time in culture by secreting fibronectin to which integrins can subsequently bind. Together, these results indicated that the secretion of fibronectin and subsequent formation of focal adhesions in cells cultured on BSA after 5 h of static culture or fluid shear stress correlates with the induction of COX-2 in response to fluid shear stress.

RGDS peptide treatment inhibits focal adhesion formation on fibronectin even after 5 h of static culture or fluid shear stress.   To verify that treatment with soluble RGDS peptide inhibited the formation of focal adhesions on fibronectin both before and after 5 h of fluid shear stress, we visualized vinculin-positive focal adhesions by immunofluorescence microscopy. Treatment with RGDS peptide limited vinculin-positive focal adhesion formation on fibronectin after 2 h of adhesion (Fig. 6g) and continued to inhibit focal adhesions after 5 h of static culture or fluid shear stress (Fig. 6, h and i). The effect of RGDS peptide was dramatic compared with both vehicle (PBS) (Fig. 6, a–c) and RGES peptide (Fig. 6, d–f) controls, which had prominent focal adhesions after 2 h of adhesion as well as after being subjected to 5 h of static culture or fluid shear stress. These results indicate that the reduction in both fluid shear stress-induced PGE2 release and upregulation of COX-2 protein correlates with inhibition of focal adhesions on fibronectin by soluble RGDS peptide treatment.



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  		Fig. 6. RGDS peptide treatment decreases focal adhesions after 5 h of static or fluid shear stress conditions. Osteoblasts treated with vehicle (PBS), RGES, or RGDS peptide were allowed to adhere to FN-coated slides for 2 h or 2 h plus 5 h of static or flow conditions before fixation in 4% paraformaldehyde and permeabilization in 0.2% Triton. Focal adhesions were visualized by using an anti-vinculin antibody. Vehicle (PBS)-treated control cells cultured on FN formed focal adhesions after 2 h of adhesion (a), and the focal adhesions appeared unaltered by 5 h of static culture or fluid shear stress (b and c). RGES control peptide (500 µg/ml) did not have any apparent effect on vinculin containing focal adhesions under static culture or fluid shear stress (d–f) compared with vehicle-treated control cells (a–c). RGDS peptide (500 µg/ml)-treated cells formed very few mature vinculin-positive focal adhesions after 2 h of adhesion (g) as well as after 5 h of static culture or fluid shear stress (h and i). Despite limited focal adhesion formation, MC3T3-E1 osteoblasts treated with RGDS peptide remained attached when subjected to fluid shear stress.

 
RGDS peptide treatment does not alter fluid shear stress-induced ERK phosphorylation.   Others studies demonstrated that COX-2 induction in response to fluid shear stress is partially dependent on ERK phosphorylation (39). Therefore, we also investigated whether focal adhesions were required for fluid shear stress-induced ERK phosphorylation in MC3T3-E1 osteoblasts. After 2 h of adhesion on glass slides coated with fibronectin, osteoblasts were subjected to a time course of fluid shear stress in the absence of serum, and both phosphorylated and total ERK protein levels were analyzed by Western blot. Like the induction of COX-2, significant phosphorylation of ERK did not occur until 5 h of fluid shear stress under these conditions (data not shown). Total ERK levels remained unchanged in response to 5 h of fluid shear stress (Fig. 7). Treatment with soluble RGDS peptide, which inhibited the induction of COX-2 in response to fluid shear stress, did not alter fluid shear stress-induced ERK phosphorylation after 5 h compared with both vehicle (PBS) and RGES peptide control cells (Fig. 7). This suggests that under our culture conditions activation of ERK is not dependent on focal adhesion formation.



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  		Fig. 7. Inhibition of focal adhesions on FN does not block fluid shear stress-induced ERK phosphorylation. Western blot analysis of total ERK1/2 revealed no change in the total levels of ERK1/2 in response to fluid shear stress under any conditions. Phosphorylated (p) ERK1/2 levels in MC3T3-E1 osteoblasts treated with RGDS, RGES, or vehicle (PBS) significantly increase in response to 5 h of fluid shear stress compared with the corresponding static counterpart (P < 0.05, Flow vs. Static, by Student's t-test). No difference was detected in the levels of fluid shear-induced ERK1/2 phosphorylation when cells were treated with vehicle (PBS), RGES, or RGDS peptide. Densitometric analysis of 5 experiments was performed.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
To date, there has been no direct assessment of the role of focal adhesions in fluid shear-induced PGE2 release and increased COX-2 expression in osteoblasts. Here, we directly investigated the role of focal adhesions in transduction of fluid shear stress-induced mechanical signaling leading to increased COX-2 protein expression and PGE2 release in MC3T3-E1 osteoblasts. COX-2 induction and PGE2 release are necessary for mechanically induced bone formation (6, 12), but little is known about how these pathways are regulated in response to fluid shear stress. The induction of COX-2 protein, and possibly PGE2 release, in response to fluid shear stress have only been linked to signaling pathways downstream of focal adhesions. Therefore, our results are significant because we show directly that focal adhesions promote these responses. Additionally, these results focus attention on the importance of focal adhesions in the regulation of mechanically induced bone formation.

By using two methods to limit the formation of focal adhesions, we determined for the first time that fibronectin-induced formation of focal adhesions promotes fluid shear stress-induced PGE2 release and upregulation of COX-2 protein. Culturing of osteoblasts on BSA inhibited the formation of focal adhesions after 2 h of culture; however, after 5 h of static culture or fluid shear stress, osteoblasts overcame this inhibition and secreted fibronectin and formed focal adhesions. Although the secretion of fibronectin and formation of focal adhesion on BSA allowed for the induction of COX-2 after 5 h of fluid shear stress, it was insufficient for the normal PGE2 response. Perhaps with longer periods of fluid shear stress the normal fluid shear stress-induced PGE2 response would be achieved. In contrast to cell cultured on BSA, focal adhesion formation was dramatically reduced both before and after 5 h of static culture or fluid shear stress when cells were treated with soluble RGDS peptide. In some studies, soluble RGDS peptide has been shown to bind to {alpha}v{beta}3-, {alpha}5{beta}1-, and {alpha}v{beta}1-integrins and to induce an active conformation (4); however, we did not see any effect of RGDS treatment on static COX-2 levels or PGE2 release compared with vehicle-treated controls or RGES peptide-treated cells. Together, these results suggest that treatment with soluble RGDS peptide did not directly activate integrins but did inhibit clustering of integrins into focal adhesions. Therefore, the significant reduction in fluid shear stress-induced COX-2 protein levels and PGE2 release in cells treated with RGDS peptide can be more specifically attributed to the limited formation of focal adhesion on fibronectin in osteoblasts.

Wadhwa et al. (39) demonstrated that fluid shear stress-induced upregulation of COX-2 was partially dependent on the phosphorylation of ERK. In our studies, we found that fluid shear stress-induced COX-2 protein, but not ERK phosphorylation, was inhibited by blocking the formation of focal adhesions with soluble RGDS peptide. Others have also demonstrated that RGDS peptide treatments, used to block integrin-matrix interactions, did not block fluid shear stress-induced ERK phosphorylation in osteoblasts (40). Together, these results suggest that other non-focal-adhesion-mediated mechanosensors, such as receptor tyrosine kinases (5), G-protein-coupled receptors (15), the glycocaylx (11, 25), or ion channels (28), may be involved in the activation of ERK.

Studies on fluid shear stress-induced mechanotransduction have used different levels of protein in the media during shear stress, including no added protein (20), 0.05% serum (39), and 2% serum (10). We found that, under our experimental conditions, cells responded similarly to fluid shear stress conditions regardless of whether the media contained added protein. In summary, we found that the formation of only a few small focal adhesions was sufficient to allow osteoblasts to adhere to the substrate and remain attached during exposure to fluid shear stress. Although osteoblasts with limited focal adhesion formation were able to release some PGE2 in response to fluid shear stress, the magnitude of the response was significantly decreased compared with cells that formed robust focal adhesions. Limiting focal adhesions and blocking integrin clustering on fibronectin with soluble RGDS peptide significantly inhibited both COX-2 induction and PGE2 release in response to fluid shear stress. The results of this study support a role for the formation of focal adhesions in the transduction of fluid shear stress-induced mechanical signaling resulting in the release of PGE2 and induction of COX-2 protein in MC3T3-E1 osteoblasts.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants AR-45218 and AR-049728 and National Aeronautics and Space Administration (NASA) Grant NAG2-1606 (awarded to F. M. Pavalko). S. M. Ponik was supported by NASA Grant NGT5-50291.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Dr. Suzanne M. Norvell for helpful suggestions and critical evaluation of the study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. M. Pavalko, Dept. of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202 (E-mail: fpavalko{at}iupui.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Alenghat FJ and Ingber DE. Mechanotransduction: all signals point to cytoskeleton, matrix, and integrins. Sci STKE 2002: PE6, 2002.
  2. Bakker AD, Soejima K, Klein-Nulend J, and Burger EH. The production of nitric oxide and prostaglandin E(2) by primary bone cells is shear stress dependent. J Biomech 34: 671–677, 2001.[CrossRef][Web of Science][Medline]
  3. Burridge K and Fath K. Focal contacts: transmembrane links between the extracellular matrix and the cytoskeleton. Bioessays 10: 104–108, 1989.[CrossRef][Web of Science][Medline]
  4. Campbell S, Otis M, Cote M, Gallo-Payet N, and Payet MD. Connection between integrins and cell activation in rat adrenal glomerulosa cells: a role for Arg-Gly-Asp peptide in the activation of the p42/p44(mapk) pathway and intracellular calcium. Endocrinology 144: 1486–1495, 2003.[Abstract/Free Full Text]
  5. Chen KD, Li YS, Kim M, Li S, Yuan S, Chien S, and Shyy JY. Mechanotransduction in response to shear stress. Roles of receptor tyrosine kinases, integrins, and Shc. J Biol Chem 274: 18393–18400, 1999.[Abstract/Free Full Text]
  6. Chow JW and Chambers TJ. Indomethacin has distinct early and late actions on bone formation induced by mechanical stimulation. Am J Physiol Endocrinol Metab 267: E287–E292, 1994.[Abstract/Free Full Text]
  7. Davies PF, Robotewskyj A, and Griem ML. Quantitative studies of endothelial cell adhesion. Directional remodeling of focal adhesion sites in response to flow forces. J Clin Invest 93: 2031–2038, 1994.[Web of Science][Medline]
  8. Dillaman RM. Movement of ferritin in the 2-day-old chick femur. Anat Rec 209: 445–453, 1984.[CrossRef][Medline]
  9. Dillaman RM, Roer RD, and Gay DM. Fluid movement in bone: theoretical and empirical. J Biomech 24: 163–177, 1991.[Web of Science][Medline]
  10. Donahue SW, Jacobs CR, and Donahue HJ. Flow-induced calcium oscillations in rat osteoblasts are age, loading frequency, and shear stress dependent. Am J Physiol Cell Physiol 281: C1635–C1641, 2001.[Abstract/Free Full Text]
  11. Florian JA, Kosky JR, Ainslie K, Pang Z, Dull RO, and Tarbell JM. Heparan sulfate proteoglycan is a mechanosensor on endothelial cells. Circ Res 93: e136–142, 2003.[Abstract/Free Full Text]
  12. Forwood MR. Inducible cyclo-oxygenase (COX-2) mediates the induction of bone formation by mechanical loading in vivo. J Bone Miner Res 11: 1688–1693, 1996.[Web of Science][Medline]
  13. Frangos JA, Eskin SG, McIntire LV, and Ives CL. Flow effects on prostacyclin production by cultured human endothelial cells. Science 227: 1477–1479, 1985.[Abstract/Free Full Text]
  14. Geiger B and Bershadsky A. Exploring the neighborhood: adhesion-coupled cell mechanosensors. Cell 110: 139–142, 2002.[CrossRef][Web of Science][Medline]
  15. Gudi SR, Clark CB, and Frangos JA. Fluid flow rapidly activates G proteins in human endothelial cells. Involvement of G proteins in mechanochemical signal transduction. Circ Res 79: 834–839, 1996.[Abstract/Free Full Text]
  16. Hert J, Liskova M, and Landrgot B. Influence of the long-term, continuous bending on the bone. An experimental study on the tibia of the rabbit. Folia Morphol (Praha) 17: 389–399, 1969.
  17. Hu X, Adamson RH, Liu B, Curry FE, and Weinbaum S. Starling forces that oppose filtration after tissue oncotic pressure is increased. Am J Physiol Heart Circ Physiol 279: H1724–H1736, 2000.[Abstract/Free Full Text]
  18. Ingber DE. Tensegrity: the architectural basis of cellular mechanotransduction. Annu Rev Physiol 59: 575–599, 1997.[CrossRef][Web of Science][Medline]
  19. Jalali S, del Pozo MA, Chen K, Miao H, Li Y, Schwartz MA, Shyy JY, and Chien S. Integrin-mediated mechanotransduction requires its dynamic interaction with specific extracellular matrix (ECM) ligands. Proc Natl Acad Sci USA 98: 1042–1046, 2001.[Abstract/Free Full Text]
  20. Jiang GL, White CR, Stevens HY, and Frangos JA. Temporal gradients in shear stimulate osteoblastic proliferation via ERK1/2 and retinoblastoma protein. Am J Physiol Endocrinol Metab 283: E383–E389, 2002.[Abstract/Free Full Text]
  21. Klein-Nulend J, Burger EH, Semeins CM, Raisz LG, and Pilbeam CC. Pulsating fluid flow stimulates prostaglandin release and inducible prostaglandin G/H synthase mRNA expression in primary mouse bone cells. J Bone Miner Res 12: 45–51, 1997.[CrossRef][Medline]
  22. Labrador V, Chen KD, Li YS, Muller S, Stoltz JF, and Chien S. Interactions of mechanotransduction pathways. Biorheology 40: 47–52, 2003.[Web of Science][Medline]
  23. Lanyon LE. Functional strain as a determinant for bone remodeling. Calcif Tissue Int 36, Suppl 1: S56–S61, 1984.
  24. Li S, Kim M, Hu YL, Jalali S, Schlaepfer DD, Hunter T, Chien S, and Shyy JY. Fluid shear stress activation of focal adhesion kinase. Linking to mitogen-activated protein kinases. J Biol Chem 272: 30455–30462, 1997.[Abstract/Free Full Text]
  25. Mochizuki S, Vink H, Hiramatsu O, Kajita T, Shigeto F, Spaan JA, and Kajiya F. Role of hyaluronic acid glycosaminoglycans in shear-induced endothelium-derived nitric oxide release. Am J Physiol Heart Circ Physiol 285: H722–H726, 2003.[Abstract/Free Full Text]
  26. Montgomery RJ, Sutker BD, Bronk JT, Smith SR, and Kelly PJ. Interstitial fluid flow in cortical bone. Microvasc Res 35: 295–307, 1988.[CrossRef][Web of Science][Medline]
  27. Norvell SM and Green KJ. contributions of extracellular and intracellular domains of full length and chimeric cadherin molecules to junction assembly in epithelial cells. J Cell Sci 111: 1305–1318, 1998.[Abstract]
  28. Olesen SP, Clapham DE, and Davies PF. Haemodynamic shear stress activates a K+ current in vascular endothelial cells. Nature 331: 168–170, 1988.[CrossRef][Medline]
  29. Pankov R and Yamada KM. Fibronectin at a glance. J Cell Sci 115: 3861–3863, 2002.[Free Full Text]
  30. Pavalko FM, Chen NX, Turner CH, Burr DB, Atkinson S, Hsieh YF, Qiu J, and Duncan RL. Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions. Am J Physiol Cell Physiol 275: C1591–C1601, 1998.[Abstract/Free Full Text]
  31. Reich KM and Frangos JA. Effect of flow on prostaglandin E2 and inositol trisphosphate levels in osteoblasts. Am J Physiol Cell Physiol 261: C428–C432, 1991.[Abstract/Free Full Text]
  32. Reich KM, McAllister TN, Gudi S, and Frangos JA. Activation of G proteins mediates flow-induced prostaglandin E2 production in osteoblasts. Endocrinology 138: 1014–1018, 1997.[Abstract/Free Full Text]
  33. Rubin CT and Lanyon LE. Regulation of bone mass by mechanical strain magnitude. Calcif Tissue Int 37: 411–417, 1985.[Web of Science][Medline]
  34. Salter DM, Robb JE, and Wright MO. Electrophysiological responses of human bone cells to mechanical stimulation: evidence for specific integrin function in mechanotransduction. J Bone Miner Res 12: 1133–1141, 1997.[CrossRef][Web of Science][Medline]
  35. Sheffield JB, Graff D, and Li HP. A solid-phase method for the quantitation of protein in the presence of sodium dodecyl sulfate and other interfering substances. Anal Biochem 166: 49–54, 1987.[CrossRef][Web of Science][Medline]
  36. Shyy JY and Chien S. Role of integrins in cellular responses to mechanical stress and adhesion. Curr Opin Cell Biol 9: 707–713, 1997.[CrossRef][Web of Science][Medline]
  37. Smalt R, Mitchell FT, Howard RL, and Chambers TJ. Mechanotransduction in bone cells: induction of nitric oxide and prostaglandin synthesis by fluid shear stress, but not by mechanical strain. Adv Exp Med Biol 433: 311–314, 1997.[Web of Science][Medline]
  38. Turner CH, Forwood MR, and Otter MW. Mechanotransduction in bone: do bone cells act as sensors of fluid flow? FASEB J 8: 875–878, 1994.[Abstract]
  39. Wadhwa S, Godwin SL, Peterson DR, Epstein MA, Raisz LG, and Pilbeam CC. Fluid flow induction of cyclo-oxygenase 2 gene expression in osteoblasts is dependent on an extracellular signal-regulated kinase signaling pathway. J Bone Miner Res 17: 266–274, 2002.[CrossRef][Web of Science][Medline]
  40. Weyts FA, Li YS, van Leeuwen J, Weinans H, and Chien S. ERK activation and alpha v beta 3 integrin signaling through Shc recruitment in response to mechanical stimulation in human osteoblasts. J Cell Biochem 87: 85–92, 2002.[Web of Science][Medline]



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