ATP is rapidly released from osteoblasts in response to mechanical load. We examined the mechanisms involved in this release and established that shear-induced ATP release was mediated through vesicular fusion and was dependent on Ca 2+ entry into the cell through L-type voltage-sensitive Ca 2+ channels. Degradation of secreted ATP by apyrase prevented shear-induced PGE 2 release.Introduction: Fluid shear induces a rapid rise in intracellular calcium ([Ca 2+ ] i ) in osteoblasts that mediates many of the cellular responses associated with mechanotransduction in bone. A potential mechanism for this increase in [Ca 2+ ] i is the activation of purinergic (P2) receptors resulting from shear-induced extracellular release of ATP. This study was designed to determine the effects of fluid shear on ATP release and the possible mechanisms associated with this release. Materials and Methods: MC3T3-E1 preosteoblasts were plated on type I collagen, allowed to proliferate to 90% confluency, and subjected to 12 dynes/cm 2 laminar fluid flow using a parallel plate flow chamber. ATP release into the flow media was measured using a luciferin/luciferase assay. Inhibitors of channels, gap junctional intercellular communication (GJIC), and vesicular formation were added before shear and maintained in the flow medium for the duration of the experiment. Results and Conclusions: Fluid shear produced a transient increase in ATP release compared with static MC3T3-E1 cells (59.8 ± 15.7 versus 6.2 ± 1.8 nM, respectively), peaking within 1 minute of onset. Inhibition of calcium entry through the L-type voltage-sensitive Ca 2+ channel (L-VSCC) with nifedipine or verapamil significantly attenuated shear-induced ATP release. Channel inhibition had no effect on basal ATP release in static cells. Ca 2+ -dependent ATP release in response to shear seemed to result from vesicular release and not through gap hemichannels. Vesicle disruption with N-ethylmaleimide, brefeldin A, or monensin prevented increases in flow-induced ATP release, whereas inhibition of gap hemichannels with either 18␣-glycyrrhetinic acid or 18␤-glycyrrhetinic acid did not. Degradation of extracellular ATP with apyrase prevented shearinduced increases in prostaglandin E 2 (PGE 2 ) release. These data suggest a time line of mechanotransduction wherein fluid shear activates L-VSCCs to promote Ca 2+ entry that, in turn, stimulates vesicular ATP release. Furthermore, these data suggest that P2 receptor activation by secreted ATP mediates flow-induced prostaglandin release.

To determine the role of Ca 2+ signaling in activation of the Mitogen-Activated Protein Kinase (MAPK) pathway, we subjected MC3T3-E1 pre-osteoblastic cells to inhibitors of Ca 2+ signaling during application of fluid shear stress (FSS). FSS only activated ERK1/2, rapidly inducing phosphorylation within 5 minutes of the onset of shear. Phosphorylation of ERK1/2 (pERK1/2) was significantly reduced when Ca 2+ i was chelated with BAPTA or when Ca 2+ was removed from the flow media. Inhibition of both the L-type voltage-sensitive Ca 2+ channel and the mechanosensitive cation-selective channel blocked FSS-induced pERK1/2. Inhibition of phospholipase C with U73122 significantly reduced pERK1/2. This inhibition did not result from block of intracellular Ca 2+ release, but a loss of PKC activation. Recent data suggests a role of ATP release and purinergic receptor activation in mechanotransduction. Apyrase-mediated hydrolysis of extracellular ATP completely blocked FSS-induced phosphorylation of ERK1/2, while addition of exogenous ATP to static cells mimicked the effects of FSS on pERK1/2. Two P2 receptors, P2Y 2 and P2X 7 , have been associated with the anabolic responses of bone to mechanical loading. Using both iRNA techniques and primary osteoblasts isolated from P2X 7 knockout mice, we found that the P2X 7 , but not the P2Y 2 , purinergic receptor was involved in ERK1/2 activation under FSS. These data suggest that FSS-induced ERK1/2 phosphorylation requires Ca 2+ -dependent ATP release, however both increased Ca 2+ i and PKC activation are needed for complete activation. Further, this ATP-dependent ERK1/2 phosphorylation is mediated through P2X 7 , but not P2Y 2 , purinergic receptors.

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Fluid shear stress regulates gene expression in osteoblasts, in part by activation of the transcription factor NF-κB. We examined whether this process was under control of purinoceptor activation. MC3T3-E1 osteoblasts under static conditions expressed the NF-κB inhibitory protein IκBα and exhibited cytosolic localization of NF-κB. Under fluid shear stress, IκBα levels decreased, and concomitant nuclear localization of NF-κB was observed. Cells exposed to fluid shear stress in ATP-depleted medium exhibited no significant reduction in IκBα, and NF-κB remained within the cytosol. Similar results were found using oxidized ATP or Brilliant Blue G, P2X 7 receptor antagonists, indicating that the P2X 7 receptor is responsible for fluid shear-stress-induced IκBα degradation and nuclear accumulation of NF-κB. Pharmacologic blockage of the P2Y6 receptor also prevented shear-induced IκBα degradation. These phenomena involved neither ERK1/2 signaling nor autocrine activation by P2X 7 -generated lysophosphatidic acid. Our results suggest that fluid shear stress regulates NF-κB activity through the P2Y 6 and P2X 7 receptor.