Activation of extracellular-signal regulated kinase (ERK1/2) by fluid shear is Ca2+- and ATP-dependent in MC3T3-E1 osteoblasts

Abstract

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.

Expression, signaling, and function of P2X7 receptors in bone

“…The phosphorylation of ERK1/2 required ATP release, and appeared to be dependent on both elevation of [Ca 2+ ] i and activation of protein kinase C (PKC) [34]. In summary, P2X7 receptors in cells of the osteoblast lineage couple to multiple signaling pathways.…”

“…Most recently, P2X7 receptors have been shown to mediate extracellular signal-regulated kinase (ERK) 1/2 activation by fluid shear stress in an osteoblast-like cell line [34,40]. The phosphorylation of ERK1/2 required ATP release, and appeared to be dependent on both elevation of [Ca 2+ ] i and activation of protein kinase C (PKC) [34].…”

The role of purinergic signalling in the musculoskeletal system

“…Furthermore, the P2X7 receptor is thought to mediate the ERK1/2 activation, NF-κB translocation and PGE2 synthesis caused by fluid shear stress in osteoblast-like cells (Genetos et al, 2005;Liu et al, 2008). Other suggested roles for the P2X7 receptor in osteoblasts include the regulation of metabolic acid production (Grol et al, 2012) and callus remodelling during fracture repair (Li et al, 2009).…”

The P2Y13 receptor regulates extracellular ATP metabolism and the osteogenic response to mechanical loading

“…(10) At the cellular level, mechanical loading-induced osteogenic response is initiated via the release of intracellular molecules such as nitric oxide and prostaglandins (PG), which are anabolic to osteoblasts. (11,12) Mechanical stimuli can also induce extracellular ATP release from a variety of cells, including osteoblasts. (13)(14)(15) This mechanism is now widely believed to be one of the transduction pathways by which mechanical stimulation initiates a cellular response.…”