Mechanical loading by fluid shear stress enhances IGF-1 receptor signaling in osteoblasts in a PKCzeta-dependent manner

Maintenance of optimal bone physiology requires the coordinated activity of osteoclasts that resorb old bone and osteoblasts that deposit new bone. Mechanical loading of bone and the resulting movement of interstitial fluid within the spaces surrounding bone cells is thought to play a key role is maintaining optimal bone mass. One way in which fluid movement may promote bone formation is by enhancing osteoblast survival. We have shown previously that application of fluid flow to osteoblasts in vitro confers a protective effect by inhibiting osteoblast apoptosis (Pavalko et al., 2003, J. Cell Physiol., 194: 194-205). To investigate the cellular mechanisms that regulate the response of osteoblasts to fluid shear stress, we have examined the possible interaction between fluid flow and growth factors in MC3T3-E1 osteoblast-like cells. We found that insulin-like growth factor-I (IGF-I) was significantly more effective at preventing TNF-alpha-induced apoptosis when cells were first subjected to mechanical loading by exposure to either unidirectional or oscillatory fluid flow compared to cells that were maintained in static culture. Additionally, downstream signaling in response to treatment with IGF-I, including ERK and Akt activation, was enhanced in cells that were subjected to fluid flow, compared to cells maintained in static culture. Furthermore, we found that PKC activity is essential for fluid shear stress sensitization of IGF-IR, since a specific inhibitor of PCKzeta function blocked the flow-enhanced IGF-I-activated Akt and ERK phosphorylation. Together, our results suggest that fluid shear stress may regulate IGF-I signaling in osteoblasts in a PKC-zeta-dependent manner.     

Protein kinase G and focal adhesion kinase converge on Src/Akt/β-catenin signaling module in osteoblast mechanotransduction

Mechanical loading of bone induces interstitial fluid flow, leading to fluid shear stress (FSS) of osteoblasts. FSS rapidly increases the intracellular calcium concentration ([Ca(2+)]) and nitric oxide (NO) synthesis in osteoblasts and activates the protein kinase Akt. Activated Akt stimulates osteoblast proliferation and survival, but the mechanism(s) leading to Akt activation is not well defined. Using pharmacological and genetic approaches in primary human and mouse osteoblasts and mouse MC3T3 osteoblast-like cells, we found that Akt activation by FSS occurred through two parallel pathways; one required calcium stimulation of NO synthase and NO/cGMP/protein kinase G II-dependent activation of Src, and the other required calcium activation of FAK and Src, independent of NO. Both pathways cooperated to increase PI3K-dependent Akt phosphorylation and were necessary for FSS to induce nuclear translocation of β-catenin, c-fos, and cox-2 gene expression and osteoblast proliferation. These data explain how mechanical stimulation of osteoblasts leads to increased signaling through a growth regulatory pathway essential for maintaining skeletal integrity.     

Application of multiple forms of mechanical loading to human osteoblasts reveals increased ATP release in response to fluid flow in 3D cultures and differential regulation of immediate early genes

ATP is actively released into the extracellular environment from a variety of cell types in response to mechanical stimuli. This is particularly true in bone where mechanically induced ATP release leads to immediate early gene activation to regulate bone remodelling; however there is no consensus as to which mechanical stimuli stimulate osteoblasts the most. To elucidate which specific type(s) of mechanical stimuli induce ATP release and gene activation in human osteoblasts, we performed an array of experiments using different mechanical stimuli applied to both monolayer and 3D cultures of the same osteoblast cell type, SaOS-2. ATP release from osteoblasts cultured in monolayer significantly increased in response to turbulent fluid flow, laminar fluid flow and substrate strain. No significant change in ATP release could be detected in 3D osteoblast cultures in response to cyclic or static compressive loading of osteoblast-seeded scaffolds, whilst turbulent fluid flow increased ATP release from 3D cultures of osteoblasts to a greater degree than observed in monolayer cultures. Cox-2 expression quantified using real time PCR was significantly lower in cells subjected to turbulent fluid flow whereas c-fos expression was significantly higher in cells subjected to strain. Load-induced signalling via c-fos was further investigated using a SaOS-2 c-fos luciferase reporter cell line and increased in response to substrate strain and turbulent fluid flow in both monolayer and 3D, with no significant change in response to laminar fluid flow or 3D compressive loading. The results of this study demonstrate for the first time strain-induced ATP release from osteoblasts and that turbulent fluid flow in 3D up regulates the signals required for bone remodelling.     

Response of osteoblasts to low fluid shear stress is time dependent

The process of mechanotransduction of bone, the conversion of a mechanical stimulus into a biochemical response, is known to occur in osteoblasts in response to fluid shear stress. In order to understand the reaction of osteoblasts to various times of flow perfusion, osteoblasts were seeded on three-dimensional scaffolds, and cultured in the following conditions: continuous flow perfusion, intermittent flow perfusion, and static condition. We collected samples on day 4, 8 and 12 for analysis. Osteoblast proliferation was demonstrated by cell proliferation and scanning electron microscopy assay. Additionally, the expression of known markers of differentiation, including alkaline phosphatase and osteocalcin, were tested by qRT-PCR and alkaline phosphatase activity assay, and the deposition of calcium was used as an indicator of mineralization demonstrated by calcium content assay. The results supported that low fluid shear stress plays an important role in the activation of osteoblasts: enhance cell proliferation, increase calcium deposition, and promote the expression of osteoblastic markers. Furthermore, the continuous flow perfusion is a more favorable environment for the initiation of osteoblast activity compared with intermittent flow perfusion. Therefore, the force and time of fluid shear stress are important parameters for osteoblast activation.     

Osteoblasts and osteocytes respond differently to oscillatory and unidirectional fluid flow profiles

Bone cells subjected to mechanical loading by fluid shear stress undergo significant architectural and biochemical changes. The models of shear stress used to analyze the effects of loading bone cells in vitro include both oscillatory and unidirectional fluid shear profiles. Although the fluid flow profile experienced by cells within bone is most likely oscillatory in nature, to date there have been few direct comparisons of how bone cells respond to these two fluid flow profiles. In this study we evaluated morphologic and biochemical responses to a time course of unidirectional and oscillatory fluid flow in two commonly used bone cell lines, MC3T3-E1 osteoblasts and MLO-Y4 osteocytes. We determined that stress fibers formed and aligned within osteoblasts after 1 h of unidirectional fluid flow, but this response was not observed until greater than 5 h of oscillatory fluid flow. Despite the delay in stress fiber formation, oscillatory and unidirectional fluid flow profiles elicited similar temporal effects on the induction of both cyclooxygenase-2 (Cox-2) and osteopontin protein expression in osteoblasts. Interestingly, MLO-Y4 osteocytes formed organized stress fibers after exposure to 24 h of unidirectional shear stress, while the number of dendritic processes per cell increased along with Cox-2 protein levels after 24 h of oscillatory shear stress. Despite these differences, both flow profiles significantly altered osteopontin levels in MLO-Y4 osteocytes. Together these results demonstrate that the profile of fluid shear can induce significantly different responses from osteoblasts and osteocytes.     

Cyclic fluid shear stress promotes osteoblastic cells proliferation through ERK5 signaling pathway

Fluid shear stress plays an important role in bone remodeling, however, the mechanism of mechanotransduction in bone tissue remains unclear. Recently, ERK5 has been found to be involved in multiple cellular processes. This study was designed to investigate the potential involvement of ERK5 in the proliferative response of osteoblastic cells to cyclic fluid shear stress. We reported here that cyclic fluid shear stress promoted ERK5 phosphorylation in MC3T3-E1 cells. Inhibition of ERK5 phosphorylation attenuated the increased expression of AP-1 and cyclin D1 and cell proliferation induced by cyclic fluid flow, but promoted p-16 expression. Further more, we found that cyclic fluid shear stress was a better stimuli for ERK5 activation and cyclin D1 expression compared with continuous fluid shear stress. Moreover, the pharmacological ERK5 inhibitor, BIX02189, which inhibited ERK5 phosphorylation in a time-dependent manner and the suppression lasted for at least 4 h. Taken together, we demonstrate that ERK5/AP-1/cyclin D1 pathway is involved in the mechanism of osteoblasts proliferation induced by cyclic fluid shear stress, which is superior in promoting cellular proliferation compared with continuous fluid shear stress.     

Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway.     

Non-overlapping functions for Pyk2 and FAK in osteoblasts during fluid shear stress-induced mechanotransduction

Mechanotransduction, the process by which cells convert external mechanical stimuli such as fluid shear stress (FSS) into biochemical changes, plays a critical role in maintenance of the skeleton. We have proposed that mechanical stimulation by FSS across the surfaces of bone cells results in formation of unique signaling complexes called mechanosomes that are launched from sites of adhesion with the extracellular matrix and with other bone cells [1]. Deformation of adhesion complexes at the cell membrane ultimately results in alteration of target gene expression. Recently, we reported that focal adhesion kinase (FAK) functions as a part of a mechanosome complex that is required for FSS-induced mechanotransduction in bone cells. This study extends this work to examine the role of a second member of the FAK family of non-receptor protein tyrosine kinases, proline-rich tyrosine kinase 2 (Pyk2), and determine its role during osteoblast mechanotransduction. We use osteoblasts harvested from mice as our model system in this study and compared the contributions of Pyk2 and FAK during FSS induced mechanotransduction in osteoblasts. We exposed Pyk2(+/+) and Pyk2(-/-) primary calvarial osteoblasts to short period of oscillatory fluid flow and analyzed downstream activation of ERK1/2, and expression of c-fos, cyclooxygenase-2 and osteopontin. Unlike FAK, Pyk2 was not required for fluid flow-induced mechanotransduction as there was no significant difference in the response of Pyk2(+/+) and Pyk2(-/-) osteoblasts to short periods of fluid flow (FF). In contrast, and as predicted, FAK(-/-) osteoblasts were unable to respond to FF. These data indicate that FAK and Pyk2 have distinct, non-redundant functions in launching mechanical signals during osteoblast mechanotransduction. Additionally, we compared two methods of generating FF in both cell types, oscillatory pump method and another orbital platform method. We determined that both methods of generating FF induced similar responses in both primary calvarial osteoblasts and immortalized calvarial osteoblasts.     

Osteoblasts respond to pulsatile fluid flow with short-term increases in PGE(2) but no change in mineralization.

Although there is no consensus as to the precise nature of the mechanostimulatory signals imparted to the bone cells during remodeling, it has been postulated that deformation-induced fluid flow plays a role in the mechanotransduction pathway. In vitro, osteoblasts respond to fluid shear stress with an increase in PGE(2) production; however, the long-term effects of fluid shear stress on cell proliferation and differentiation have not been examined. The goal of this study was to apply continuous pulsatile fluid shear stresses to osteoblasts and determine whether the initial production of PGE(2) is associated with long-term biochemical changes. The acute response of bone cells to a pulsatile fluid shear stress (0.6 +/- 0.5 Pa, 3.0 Hz) was characterized by a transient fourfold increase in PGE(2) production. After 7 days of static culture (0 dyn/cm(2)) or low (0.06 +/- 0.05 Pa, 0.3 Hz) or high (0.6 +/- 0.5 Pa, 3.0 Hz) levels of pulsatile fluid shear stress, the bone cells responded with an 83% average increase in cell number, but no statistical difference (P > 0.53) between the groups was observed. Alkaline phosphatase activity per cell decreased in the static cultures but not in the low- or high-flow groups. Mineralization was also unaffected by the different levels of applied shear stress. Our results indicate that short-term changes in PGE(2) levels caused by pulsatile fluid flow are not associated with long-term changes in proliferation or mineralization of bone cells.     

Evaluation of different culture techniques of osteoblasts on 3D scaffolds

Bones adjust their structure to withstand the mechanical demands they experience. It is suggested that flow-derived shear stress may be the most significant and primary mediator of mechanical stimulation. In this study, we designed and fabricated a fluid flow cell culture system that can load shear stress onto cells cultured on 3D scaffolds. We evaluated the effect of different culture techniques, namely, (1) continuous perfusion fluid flow, (2) intermittent perfusion fluid flow, and (3) static condition, on the proliferation of osteoblasts seeded on partially deproteinized bones. The flow rate was set at 1 ml/min for all the cells cultured using flow perfusion and the experiment was conducted for 12 days. Scanning electron microscopy analysis indicated an increase in cell proliferation for scaffolds subjected to fluid shear stress. In addition, the long axes of these cells lengthened along the flowing fluid direction. Continuous perfusion significantly enhanced cell proliferation compared to either intermittent perfusion or static condition. All the results demonstrated that fluid shear stress is able to enhance the proliferation of cells and change the form of cells.     

E11/gp38 selective expression in osteocytes: regulation by mechanical strain and role in dendrite elongation

Within mineralized bone, osteocytes form dendritic processes that travel through canaliculi to make contact with other osteocytes and cells on the bone surface. This three-dimensional syncytium is thought to be necessary to maintain viability, cell-to-cell communication, and mechanosensation. E11/gp38 is the earliest osteocyte-selective protein to be expressed as the osteoblast differentiates into an osteoid cell or osteocyte, first appearing on the forming dendritic processes of these cells. Bone extracts contain large amounts of E11, but immunostaining only shows its presence in early osteocytes compared to more deeply embedded cells, suggesting epitope masking by mineral. Freshly isolated primary osteoblasts are negative for E11 expression but begin to express this protein in culture, and expression increases with time, suggesting differentiation into the osteocyte phenotype. Osteoblast-like cell lines 2T3 and Oct-1 also show increased expression of E11 with differentiation and mineralization. E11 is highly expressed in MLO-Y4 osteocyte-like cells compared to osteoblast cell lines and primary osteoblasts. Differentiated, mineralized 2T3 cells and MLO-Y4 cells subjected to fluid flow shear stress show an increase in mRNA for E11. MLO-Y4 cells show an increase in dendricity and elongation of dendrites in response to shear stress that is blocked by small interfering RNA specific to E11. In vivo, E11 expression is also increased by a mechanical load, not only in osteocytes near the bone surface but also in osteocytes more deeply embedded in bone. Maximal expression is observed not in regions of maximal strain but in a region of potential bone remodeling, suggesting that dendrite elongation may be occurring during this process. These data suggest that osteocytes may be able to extend their cellular processes after embedment in mineralized matrix and have implications for osteocytic modification of their microenvironment.     

Neuropeptide Y is expressed by osteocytes and can inhibit osteoblastic activity

Osteocytes are the most abundant osteoblast lineage cells within the bone matrix. They respond to mechanical stimulation and can participate in the release of regulatory proteins that can modulate the activity of other bone cells. We hypothesize that neuropeptide Y (NPY), a neurotransmitter with regulatory functions in bone formation, is produced by osteocytes and can affect osteoblast activity. To study the expression of NPY by the osteoblast lineage cells, we utilized transgenic mouse models in which we can identify and isolate populations of osteoblasts and osteocytes. The Col2.3GFP transgene is active in osteoblasts and osteocytes, while the DMP1 promoter drives green fluorescent protein (GFP) expression in osteocytes. Real‐time PCR analysis of RNA from the isolated populations of cells derived from neonatal calvaria showed higher NPY mRNA in the preosteocytes/osteocytes fraction compared to osteoblasts. NPY immunostaining confirmed the strong expression of NPY in osteocytes (DMP1GFP⁺), and lower levels in osteoblasts. In addition, the presence of NPY receptor Y1 mRNA was detected in cavaria and long bone, as well as in primary calvarial osteoblast cultures, whereas Y2 mRNA was restricted to the brain. Furthermore, NPY expression was reduced by 30–40% in primary calvarial cultures when subjected to fluid shear stress. In addition, treatment of mouse calvarial osteoblasts with exogenous NPY showed a reduction in the levels of intracellular cAMP and markers of osteoblast differentiation (osteocalcin, BSP, and DMP1). These results highlight the potential regulation of osteoblast lineage differentiation by local NPY signaling. J. Cell. Biochem. 108: 621–630, 2009. © 2009 Wiley‐Liss, Inc.     

Fluid Flow-induced Soluble Vascular Endothelial Growth Factor Isoforms Regulate Actin Adaptation in Osteoblasts

Although load-induced mechanical signals play a key role in bone formation and maintenance of bone mass and structure, the cellular mechanisms involved in the translation of these signals are still not well understood. Recent identification of a novel flow-induced mechanosignaling pathway involving VEGF in osteoblasts and the known VEGF regulation of actin reorganization in various cell types has led us to hypothesize that fluid shear stress-induced Vegf up-regulation underlies the actin cytoskeleton adaptation observed in osteoblasts during mechanotransduction. Our results show that MC3T3-E1 cells secrete significant VEGF in response to 5 h of pulsatile fluid shear stress (PFSS; 5 dynes/cm² at 1 Hz), whereas expression of VEGF receptors (VEGFR-1, VEGFR-2, or NRP1) is unaffected. These receptors, in particular VEGFR-2, participate in PFSS-induced VEGF release. Exposure to flow-conditioned medium or exogenous VEGF significantly induces stress fiber formation in osteoblasts that is comparable with PFSS-induced stress fiber formation, whereas VEGF knockdown abrogates this response to PFSS, thereby providing evidence that flow-induced VEGF release plays a role in actin polymerization. Using neutralizing antibodies against the receptors and VEGF isoforms, we found that soluble VEGFs, in particular VEGF₁₆₄, play a crucial role in transient stress fiber formation during osteoblast mechanotransduction, most likely through VEGFR-2 and NRP1. Based on these data we conclude that flow-induced VEGF release from osteoblasts regulates osteoblast actin adaptation during mechanotransduction and that VEGF paracrine signaling may provide potent cross-talk among bone cells and endothelial cells that is essential for fracture healing, bone remodeling, and osteogenesis.     

Possible involvement of phosphatidylinositol 3-kinase/Akt pathway in insulin-like growth factor-I-induced alkaline phosphatase activity in osteoblasts.

In the present study, we investigated whether Akt is involved in insulin-like growth factor-I (IGF-I)-stimulated activity of alkaline phosphatase, a marker of mature osteoblast phenotype, in osteoblast-like MC3T3-E1 cells. IGF-I induced the phosphorylation of Akt in these cells. Akt inhibitor significantly suppressed the IGF-I-stimulated alkaline phosphatase activity. The phosphorylation of Akt induced by IGF-I was reduced by the Akt inhibitor. LY294002 and wortmannin, inhibitors of phosphatidylinositol 3-kinase, significantly suppressed the IGF-I-induced alkaline phosphatase activity. The phosphorylation of Akt induced by IGF-I was markedly reduced by LY294002 and wortmannin. These results strongly suggest that phosphatidylinositol 3-kinase/Akt plays a role in the IGF-I-stimulated alkaline phosphatase activity in osteoblasts.     

The role of signaling pathways in osteoblast gravity perception

Bone loss is one of the major problems in long term spaceflight. This physiological consequence of microgravity is the rapid loss of weightbearing bone that is associated with skeletal unloading. Moreover, we have previously noted that sera deprived osteoblasts do not have a normal response to sera in microgravity. Where exercise (mechanical loading) has been shown to increase bone formation and stimulate osteoblastic function, the mechanisms underlying signal transduction of mechano-perception is yet to be fully understood. Osteoblasts have been shown to respond to mechanical stress such as fluid shear, bending, flexing and compression. The type of stress and amount of stress determine the osteoblast response Recently we have discovered that the isolated osteoblast responds to a very short pulse of g-force compression. The possible regulatory sensors include mechano-sensitive calcium channels, autrocrine responses to stress, response to FAK/integrin, alterations in the cytoskeleton as well as other known growth factor and cytokine receptors. The secondary signal may include growth factor related kinases such as ERK, p38 and JNK map kinase (MAPK) pathways. Experimental evidence suggests that normal osteoblast response to stress and sera requires normal earth gravity.     

Substrate modulation of osteoblast adhesion strength, focal adhesion kinase activation, and responsiveness to mechanical stimuli

Osteoblast interactions with extracellular matrix (ECM) proteins are known to influence many cell functions, which may ultimately affect osseointegration of implants with the host bone tissue. Some adhesion-mediated events include activation of focal adhesion kinase, and subsequent changes in the cytoskeleton and cell morphology, which may lead to changes in adhesion strength and cell responsiveness to mechanical stimuli. In this study we examined focal adhesion kinase activation (FAK), F-actin cytoskeleton reorganization, adhesion strength, and osteoblast responsiveness to fluid shear when adhered to type I collagen (ColI), glass, poly-L-lysine (PLL), fibronectin (FN), vitronectin (VN), and serum (FBS). In general, surfaces that bind cells through integrins (FN, VN, FBS) elicited the highest adhesion strength, FAK activation, and F-actin stress fiber formation after both 15 and 60 minutes of adhesion. In contrast, cells attached through non-integrin mediated means (PLL, glass) showed the lowest FAK activation, adhesion strength, and little F-actin stress fiber formation. When subjected to steady fluid shear using a parallel plate flow chamber, osteoblasts plated on FN released significantly more PGE2 compared to those on glass. In contrast, PGE2 release of osteoblasts attached to FN or glass was not different in the absence of fluid shear, suggesting that differences in binding alone are insufficient to alter PGE2 secretion. The increased adhesion strength as well as PGE2 secretion of osteoblasts adhered via integrins may be due to increased F-actin fiber formation, which leads to increased cell stiffness.     

Temporal gradients in shear stimulate osteoblastic proliferation via ERK1/2 and retinoblastoma protein

Bone cells are subject to interstitial fluid flow (IFF) driven by venous pressure and mechanical loading. Rapid dynamic changes in mechanical loading cause transient gradients in IFF. The effects of pulsatile flow (temporal gradients in fluid shear) on rat UMR106 cells and rat primary osteoblastic cells were studied. Pulsatile flow induced a 95% increase in S-phase UMR106 cells compared with static controls. In contrast, ramped steady flow stimulated only a 3% increase. Similar patterns of S-phase induction were also observed in rat primary osteoblastic cells. Pulsatile flow significantly increased relative UMR106 cell number by 37 and 62% at 1.5 and 24 h, respectively. Pulsatile flow also significantly increased extracellular signal-regulated kinase (ERK1/2) phosphorylation by 418%, whereas ramped steady flow reduced ERK1/2 activation to 17% of control. Correspondingly, retinoblastoma protein was significantly phosphorylated by pulsatile fluid flow. Inhibition of mitogen-activated protein (MAP)/ERK kinase (MEK)1/2 by U0126 (a specific MEK1/2 inhibitor) reduced shear-induced ERK1/2 phosphorylation and cell proliferation. These findings suggest that temporal gradients in fluid shear stress are potent stimuli of bone cell proliferation.     

Fluid shear stress as a mediator of osteoblast cyclic adenosine monophosphate production

Effects of interstitial fluid flow on osteoblasts were investigated. Intracellular cyclic adenosine monophosphate (cAMP) levels were monitored in cultured osteoblasts subjected to shear rates ranging from 10 to 3,500 sec-1. Cyclic AMP levels were significantly increased at all shear rates from 1 pmole/mg protein to 10-16 pmole/mg protein. Osteoblasts subjected to a shear rate of 430 sec-1 for 0.5-15 minutes exhibited elevated levels (12-fold) of intracellular cAMP, which were sustained throughout the perfusion period. Osteoblasts were three times more sensitive to flow stimulation than human umbilical vein endothelial cells and baby hamster kidney fibroblasts, which also displayed higher cAMP levels (4-fold) after exposure to flow. To distinguish streaming potential effects from shear stress effects, viscosity was increased 5-fold by addition of neutral dextran to the perfusing medium. Shear stress is a function of viscosity, and streaming potentials are not for a given shear rate. The mechanism of this cellular response to flow was shown to be shear stress dependent. Inhibition of cyclooxygenase by 20 microM ibuprofen completely inhibited the flow-dependent cAMP response, indicating the cAMP response is mediated by prostaglandins. Our results suggest that fluid flow induced by mechanical stress may be an important mediator of bone remodeling.