Mechanically stimulated osteocytes regulate osteoblastic activity via gap junctions

Abstract

The strong correlation between a bone's architectural properties and the mechanical forces that it experiences has long been attributed to the existence of a cell that not only detects mechanical load but also structurally adapts the bone matrix to counter it. One of the most likely cellular candidates for such a "mechanostat" is the osteocyte, which resides within the mineralized bone matrix and is perfectly situated to detect mechanically induced signals. However, as osteocytes can neither form nor resorb bone, it has been hypothesized that they orchestrate mechanically induced bone remodeling by coordinating the actions of cells residing on the bone surface, such as osteoblasts. To investigate this hypothesis, we developed a novel osteocyte-osteoblast coculture model that mimics in vivo systems by permitting us to expose osteocytes to physiological levels of fluid shear while shielding osteoblasts from it. Our results show that osteocytes exposed to a fluid shear rate of 4.4 dyn/cm 2 rapidly increase the alkaline phosphatase activity of the shielded osteoblasts and that osteocytic-osteoblastic physical contact is a prerequisite. Furthermore, both functional gap junctional intercellular communication and the mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 signaling pathway are essential components in the osteoblastic response to osteocyte communicated mechanical signals. By utilizing other nonosteocytic coculture models, we also show that the ability to mediate osteoblastic alkaline phosphatase levels in response to the application of fluid shear is a phenomena unique to osteocytes and is not reproduced by other mesenchymal cell types.osteocyte; osteoblast; fluid-flow; coculture; mechanical stimulation; gap junction; intercellular communication ONE OF THE major tenets of bone biology is that mechanical load perturbs interstitial fluid and that, upon detecting these perturbations, osteocytes embedded deep within mineralized bone remotely coordinate the adaptive response by directing the actions of effector cells such as bone-forming osteoblasts and bone-resorbing osteoclasts (4, 12). Although this theory is widely accepted, there is little experimental evidence that, in response to fluid shear, embedded osteocytes directly alter the cellular behavior of surface-residing osteoblasts or osteoclasts. In vivo, osteocytes pass dendritic-like cellular processes through channels in the mineralized matrix (canaliculi) and form physical connections, specifically gap junctions, with both neighboring osteocytes and surface-residing bone cells (11,23). We hypothesize that these intercellular gap junctions provide the means by which mechanically induced fluid shear signals may be communicated from osteocytes to effector cells such as osteoblasts and osteoclasts.There is abundant evidence suggesting that gap junctional intercellular communication (GJIC) contributes to mechanotransduction in the musculoskeletal system. Banes et al. (3) demonstrated that equibiaxial strain upregulates connexin 43 (C...

Osteocytes as mechanosensors in the inhibition of bone resorption due to mechanical loading

“…Note that the fluid flow to which osteocytes were exposed in this study is 2 hours. Several others studies (1,16,50,51,57) have shown that mechanical stimuli at this duration can induce many changes in markers of bone formation/resorption in bone cells. We expect that with longer flow exposure times, the bone resorption inhibition effect from mechanical loading might be further enhanced.…”

A sclerostin-based theory for strain-induced bone formation

“…Currently the inhibitory pathway via sclerostin receives a lot of attention, but there are also several studies pointing to stimulatory pathways (Heino et al 2004;Imai et al 2009;Tatsumi et al 2007;Taylor et al 2007;Vezeridis et al 2006). Tatsumi et al (2007) were able to specifically kill 70-80% of osteocytes in mice, via a toxin that did not directly affect other bone cells; although they observed a marked decrease in sclerostin mRNA, they did not observe an increase in osteoblast activity, which lead them to suggest that "it is conceivable that in addition to negative regulators such as sclerostin, one or more positive regulators are produced by osteocytes".…”

“…Tatsumi et al (2007) were able to specifically kill 70-80% of osteocytes in mice, via a toxin that did not directly affect other bone cells; although they observed a marked decrease in sclerostin mRNA, they did not observe an increase in osteoblast activity, which lead them to suggest that "it is conceivable that in addition to negative regulators such as sclerostin, one or more positive regulators are produced by osteocytes". Taylor et al (2007) co-cultured osteocytes exposed to fluid flow with osteoblasts shielded from this flow but connected to the osteocytes via gap junctions: fluid flow on osteocytes led to increased alkaline phosphatase activity in osteoblasts, but not if gap junctions were blocked. This indicates that there was a stimulatory signal reaching the osteoblasts via gap junctions.…”

“…These stimulatory signals have not been identified yet. Prostaglandins have been implicated (Chow and Chambers 1994;Burger and Klein-Nulend 1999), but their role is unclear (Suponitzky and Weinreb 1998;Taylor et al 2007), and may have to do more with regulating osteocyte gap junctions (Cheng et al 2001), than directly stimulating osteoblast activity.…”

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.…”