Osteopontin Gene Regulation by Oscillatory Fluid Flow via Intracellular Calcium Mobilization and Activation of Mitogen-activated Protein Kinase in MC3T3–E1 Osteoblasts

Abstract

Recently fluid flow has been shown to be a potent physical stimulus in the regulation of bone cell metabolism. However, most investigators have applied steady or pulsing flow profiles rather than oscillatory fluid flow, which occurs in vivo because of mechanical loading. Here oscillatory fluid flow was demonstrated to be a potentially important physical signal for loading-induced changes in bone cell metabolism. We selected three well known biological response variables including intracellular calcium (Ca

Molecular pathways mediating mechanical signaling in bone

“…Upon phosphorylation, FAK contributes to MAPK activation via interaction with c-src, Grb2, and the small GTPase Ras (Schlaepfer et al, 1997(Schlaepfer et al, , 1998(Schlaepfer et al, , 1999. This is significant since MAPK activation is also one of the effectors of oscillatory flow in bone cells (You et al, 2001) and this pathway has been observed in response to fluid flow in endothelial cells (Berk et al, 1995;Ishida et al, 1996;Li et al, 1997). It is also notable that FAK activation has been observed to cause intracellular calcium mobilization via activation of the γ1 isoform of PLC (the only SH2 containing isoform known).…”

“…Thus, straining osteoprogenitor cells can cause them to proliferate (Zhuang et al, 1996) and to secrete extracellular matrix (Harter et al, 1995). Similarly, shear stress can induce beta-catenin signaling (Norvell et al, 2004) and secretion of osteopontin (You et al, 2001). To achieve these ends, it is quite clear that multiple classic signaling pathways are activated after force application.…”

“…Numerous studies of both osteoblasts and osteocytes demonstrate that this is a consistent and robust response to both static and dynamic patterns of shear (Hung et al, 1995;Jacobs et al, 1998). Furthermore when this critical early second messenger is blocked during mechanical stimulation mechanically regulated changes in gene expression do not occur (Chen et al, 2000;You et al, 2001), although PGE 2 release in response to fluid flow occurs independent of intracellular calcium signaling (Saunders et al, 2003). This rapid increase in intracellular calcium concentration appears to occur due to phospholipase C activity and IP3 signaling resulting in release of calcium from intracellular stores in many cases.…”

“…This rapid increase in intracellular calcium concentration appears to occur due to phospholipase C activity and IP3 signaling resulting in release of calcium from intracellular stores in many cases. Interestingly, calcium-induced calcium release has been suggested in the transduction of steady fluid flow (Chen et al, 2000) but was not found to occur in response to dynamic flow (You et al, 2001) suggesting that cells may be able to distinguish temporal patterns of mechanical stimulation. Stretch is also known to stimulate the IP3 pathway in cardiomyocytes (Dassouli et al, 1993), and is probably a fairly universal response in many tissues.…”

The p38α MAPK positively regulates osteoblast function and postnatal bone acquisition

“…Previous in vitro studies using cell lines or primary osteoblasts with selective p38 inhibitors have shown that the p38 MAPK pathway regulates alkaline phosphatase activity and extracellular matrix mineralization in response to different osteogenic signals such as BMP-2 [4,5], TGFb [5], epinephrine [6], PTH [7], Wnt proteins [8] and mechanical stimuli [9]. Moreover, several reports have also indicated that the p38 pathway regulates the expression and activation of critical transcription factors implicated in osteoblastogenesis, i.e., Dlx5, Runx2, and Osx [5,[10][11][12].…”