Activation of Extracellular-signal Regulated Kinase (ERK1/2) by Fluid Shear is Ca²⁺- and ATP-dependent in MC3T3-E1 Osteoblasts

To determine the role of Ca signaling in activation of the Mitogen-Activated Protein Kinase (MAPK) pathway, we subjected MC3T3-E1 pre-osteoblastic cells to inhibitors of Ca 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 Cai was chelated with BAPTA or when Ca was removed from the flow media. Inhibition of both the L-type voltage-sensitive Ca channel and the mechanosensitive cationselective 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 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, P2Y2 and P2X7, have been associated with the anabolic NOT THE PUBLISHED VERSION; this is the author’s final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page. Bone, Vol. 42, No. 4 (April 2008): pg. 644-652. DOI. This article is © Elsevier and permission has been granted for this version to appear in e-Publications@Marquette. Elsevier does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from Elsevier. 3 responses of bone to mechanical loading. Using both iRNA techniques and primary osteoblasts isolated from P2X7 knockout mice, we found that the P2X7, but not the P2Y2, purinergic receptor was involved in ERK1/2 activation under FSS. These data suggest that FSS-induced ERK1/2 phosphorylation requires Ca-dependent ATP release, however both increased Cai and PKC activation are needed for complete activation. Further, this ATP-dependent ERK1/2 phosphorylation is mediated through P2X7, but not P2Y2, purinergic receptors.