Activation of G Proteins Mediates Flow-Induced Prostaglandin E₂Production in Osteoblasts

Abstract

Interstitial fluid flow may play a role in load-induced bone remodeling. Previously, we have shown that fluid flow stimulates osteoblast production of cAMP inositol trisphosphate (IP3), and PGE2. Flow-induced increases in cAMP and IP3 were shown to be a result of PG production. Thus, PGE2 production appears to be an important component in fluid flow induced signal transduction. In the present study, we investigated the mechanism of flow-induced PGE2 synthesis. Flow-induced a 20-fold increase in PGE2 production in osteoblasts. Increases were also observed with ALF4-(10mM) (98-fold), an activator of guanidine nucleotide-binding proteins (G proteins), and calcium ionophore A23187 (2 microM) (100-fold) in stationary cells. We then investigated whether flow stimulation is mediated by G proteins and increases in intracellular calcium. Flow-induced PGE2 production was inhibited by the G protein inhibitors GDP beta S (100 microM) and pertussis toxin (1 microgram/ml) by 83% and 72%, respectively. Chelation of extracellular calcium by EGTA (2 mM) and intracellular calcium by quin-2/AM (30 microM) blocked flow stimulation by 87% and 67%, respectively. These results suggest that G proteins and calcium play an important role in mediating mechanochemical signal transduction in osteoblasts.

Pertussis toxin‐sensitive Gαi protein and ERK‐dependent pathways mediate ultrasound promotion of osteogenic transcription in human osteoblasts1

“…Previous studies have demonstrated that G proteins were involved in strain‐activated proliferation of human endothelial cells [18]. These molecules mediated steady/transient fluid shear stress‐stimulated nitric oxide production [19,20], and flow‐induced prostaglandin E 2 production by osteoblasts [21]. Extracellular signal‐regulated kinase (ERK), a member of the mitogen‐activated protein kinase family, mediated the transduction of mechanical stimulation into intracellular signals that regulate cell proliferation and differentiation [23,24].…”

Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

“…The in vitro mechanical stimulation of bone cells by fluid flow has been reported to impact the levels of many biochemical factors including intracellular calcium (5,6), nitric oxide (4,(7)(8)(9), prostaglandin E 2 (3,4,7,8), the expression of the genes for osteopontin, cyclooxygenase-2, and c-Fos (6,(10)(11)(12) as well as other intracellular messengers and transcription factors (6,(13)(14)(15). This mechanostimulation of bone cells in vitro by fluid flow mimics the physiological response of bone cells in vivo where pressure gradients from mechanical loading of locomotion and other stressors deform the mineralized bone matrix and move extracellular fluid radially outward toward the cortex through the lacunocanalicular network (16)(17)(18)(19).…”

Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces

“…Fluid flow-induced shear stress applied to osteoblastic cells for several hours has been shown to stimulate the release of nitric oxide (5,8,9), a short-lived radical and messenger implicated in several cellular functions, and prostaglandin (4,5,8,10,11) that may have an autocrine effect on osteoblastic cells. Fluid shear has been shown to up-regulate a variety of genes, including those of osteopontin and cyclooxygenase-2, and several other transcription factors and intracellular messenger systems like cAMP, mitogen-activated protein kinase, and G proteins (12)(13)(14)(15)(16)(17). In addition, short-term fluid flow causes changes in the cell cytoskeleton by altering structural proteins and gap junctions (18)(19)(20).…”