Gap junctions and fluid flow response in MC3T3-E1 cells

Abstract

In the current study, we examined the role of gap junctions in oscillatory fluid flow-induced changes in intracellular Ca(2+) concentration and prostaglandin release in osteoblastic cells. This work was completed in MC3T3-E1 cells with intact gap junctional communication as well as in MC3T3-E1 cells rendered communication deficient through expression of a dominant-negative connexin. Our results demonstrate that MC3T3-E1 cells with intact gap junctions respond to oscillatory fluid flow with significant increases in prostaglandin E(2) (PGE(2)) release, whereas cells with diminished gap junctional communication do not. Furthermore, we found that cytosolic Ca(2+) (Ca) response was unaltered by the disruption in gap junctional communication and was not significantly different among the cell lines. Thus our results suggest that gap junctions contribute to the PGE(2) but not to the Ca response to oscillatory fluid flow. These findings implicate gap junctional intercellular communication (GJIC) in bone cell ensemble responsiveness to oscillatory fluid flow and suggest that gap junctions and GJIC play a pivotal role in mechanotransduction mechanisms in bone.

Novel early response genes in osteoblasts exposed to dynamic fluid flow

“…Consistent with previous studies (Saunders et al 2001;Donahue et al 2003b), which showed increased prostaglandin E2 (PGE 2 ) release in MC3T3-E1 cells shortly after exposure to dynamic fluid flow, our results demonstrate a 9.6-fold increase in the mRNA level of the enzyme catalysing PGE 2 production, cyclooxygenase 2 (prostaglandin-endoperoxide synthase 2; Ptgs2), at 30 min post-flow. At 1 h post-flow there was a 3.1-fold increase in Ptgs2.…”

“…A variety of cell-level biophysical signals occur due to this loading, including, but not limited to, direct cellular deformation, generation of bioelectric fields and interstitial fluid flow (Bloomfield 2001). In vitro cell culture experiments have suggested that dynamic fluid flow at levels and frequencies expected to occur due to loading is associated with numerous osteogenic effects (Jacobs et al 1998;You et al 2000You et al , 2001You et al , 2002Donahue et al 2001Donahue et al , 2003aSaunders et al 2001Saunders et al , 2003Li et al 2004;Kim et al 2006), and may be responsible in part or in full for bone mechanosensitivity (Qin et al 2003).…”

Molecular pathways mediating mechanical signaling in bone

“…This application might occur due to the larger area occupied by a cell network, and therefore larger net effect of the biophysical signal, than by individual cells. Indeed, when the communication of an ensemble of cells is interrupted, a reduced sensitivity has been observed in response to biophysical signals such as electric fields (Vander Molen et al, 2000), fluid flow (Saunders et al, 2001) or biochemical stimulation (Vander Molen et al, 1996). Interestingly, it has recently been suggested that in addition to regulating cell-cell communication, connexins can also form regulatory channels between the cell and its extracellular environment.…”

Connexin43 Potentiates Osteoblast Responsiveness to Fibroblast Growth Factor 2 via a Protein Kinase C-Delta/Runx2–dependent Mechanism

“…These conditional Cx43 knockout mice, which use a 2.3-kb collagen I promoter-driven Cre-recombinase to excise a floxed Cx43 allele in cells of the osteoblast lineage, exhibit severe osteopenia as a result of defective osteoblast function. Further, the skeletons of these animals have been shown to be refractory to the anabolic effects of intermittent PTH administration, indicating a critical role of Cx43 in coordinating cell function in response to anabolic cues.Relatively little is known about the precise molecular role of gap junctions in sensing and responding to biological cues during bone formation, though in the past decade critical insights have been gained into the importance of gap junctions in processes such as response to growth factors and hormones (Schiller et al, 1992;Chiba et al, 1994;Van der Molen et al, 1996;Civitelli et al, 1998;Schiller et al, 2001), mechanical load (Jorgensen et al, 1997(Jorgensen et al, , 2000Ziambaras et al, 1998;Romanello and D'Andrea, 2001;Saunders et al, 2001Saunders et al, , 2003Cherian et al, 2003Cherian et al, , 2005Taylor et al, 2007), bisphosphonates (Plotkin et al, 2002(Plotkin et al, , 2005, and interaction with other cells (Villars et al, 2002). Importantly, less still is known regarding the biologically relevant second messengers that osteoblast and osteocyte gap junctions communicate among cells, and many molecular mechanisms have yet to be deduced.…”