Mechanical Stimulation of Gap Junctions in Bone Osteocytes is Mediated by Prostaglandin E2

Gap junction-mediated intercellular communications are thought to transduce the effects of mechanical strain from osteocytes to cells on the bone surface to initiate remodeling. To determine whether gap junctions may co-ordinate the effects of mechanical loading, osteocyte-like MLO-Y4 cells were exposed to fluid flow-imposed shear stress. After exposure of MLO-Y4 to fluid flow, intercellular coupling increased in direct proportion to shear stress level. Interestingly, this stimulation is further enhanced during the post-stress period, indicating that released factors) is likely to be involved. The conditioned medium obtained from the fluid flow treated MLO-Y4 cells induced an increase in the number of functional gap junctions and Cx43 protein when added to non-sheer-stressed cells. Fluid flow was found to induce prostaglandin E₂ (PGE₂) release and increase cyclooxygenase 2 (COX-2) expression. When PGE₂ was depleted from the fluid flow conditioned medium, the stimulatory effect on gap junctions was significantly decreased. Addition of the COX inhibitor indomethacin partially blocked the stimulatory effects of mechanical strain on gap junctions. Together, these studies suggest that the stimulatory effect of fluid flow on gap junctions is mediated in part by de novo synthesis and release of PGE₂. Gap junctions may serve as channels for the signals generated by osteocytes in response to mechanical loading.     

Effects of mechanical strain on the function of Gap junctions in osteocytes are mediated through the prostaglandin EP2 receptor

Osteocytes embedded in the matrix of bone are thought to be mechanosensory cells that translate mechanical strain into biochemical signals that regulate bone modeling and remodeling. We have shown previously that fluid flow shear stress dramatically induces prostaglandin release and COX-2 mRNA expression in osteocyte-like MLO-Y4 cells, and that prostaglandin E2 (PGE2) released by these cells functions in an autocrine manner to regulate gap junction function and connexin 43 (Cx43) expression. Here we show that fluid flow regulates gap junctions through the PGE2 receptor EP2 activation of cAMP-dependent protein kinase A (PKA) signaling. The expression of the EP2 receptor, but not the subtypes EP1,EP3, and EP4, increased in response to fluid flow. Application of PGE2 or conditioned medium from fluid flow-treated cells to non-stressed MLO-Y4 cells increased expression of the EP2 receptor. The EP2 receptor antagonist, AH6809, suppressed the stimulatory effects of PGE2 and fluid flow-conditioned medium on the expression of the EP2 receptor, on Cx43 protein expression, and on gap junction-mediated intercellular coupling. In contrast, the EP2 receptor agonist butaprost, not the E1/E3 receptor agonist sulprostone, stimulated the expression of Cx43 and gap junction function. Fluid flow conditioned medium and PGE2 stimulated cAMP production and PKA activity suggesting that PGE2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE2 on gap junctions. These studies suggest that the EP2 receptor mediates the effects of autocrine PGE2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP2 receptor, cAMP, and PKA are critical components of the signaling cascade between mechanical strain and gap junction-mediated communication between osteocytes.     

Oscillating fluid flow activation of gap junction hemichannels induces ATP release from MLO-Y4 osteocytes

Mechanical loads are required for optimal bone mass. One mechanism whereby mechanical loads are transduced into localized cellular signals is strain-induced fluid flow through lacunae and canaliculi of bone. Gap junctions (GJs) between osteocytes and osteoblasts provides a mechanism whereby flow-induced signals are detected by osteocytes and transduced to osteoblasts. We have demonstrated the importance of GJ and gap junctional intercellular communication (GJIC) in intracellular calcium and prostaglandin E(2) (PGE(2)) increases in response to flow. Unapposed connexons, or hemichannels, are themselves functional and may constitute a novel mechanotransduction mechanism. Using MC3T3-E1 osteoblasts and MLO-Y4 osteocytes, we examined the time course and mechanism of hemichannel activation in response to fluid flow, the composition of the hemichannels, and the role of hemichannels in flow-induced ATP release. We demonstrate that fluid flow activates hemichannels in MLO-Y4, but not MC3T3-E1, through a mechanism involving protein kinase C, which induces ATP and PGE(2) release.     

Mechanical strain opens connexin 43 hemichannels in osteocytes: a novel mechanism for the release of prostaglandin

Mechanosensing bone osteocytes express large amounts of connexin (Cx)43, the component of gap junctions; yet, gap junctions are only active at the small tips of their dendritic processes, suggesting another function for Cx43. Both primary osteocytes and the osteocyte-like MLO-Y4 cells respond to fluid flow shear stress by releasing intracellular prostaglandin E2 (PGE2). Cells plated at lower densities release more PGE2 than cells plated at higher densities. This response was significantly reduced by antisense to Cx43 and by the gap junction and hemichannel inhibitors 18 beta-glycyrrhetinic acid and carbenoxolone, even in cells without physical contact, suggesting the involvement of Cx43-hemichannels. Inhibitors of other channels, such as the purinergic receptor P2X7 and the prostaglandin transporter PGT, had no effect on PGE2 release. Cell surface biotinylation analysis showed that surface expression of Cx43 was increased by shear stress. Together, these results suggest fluid flow shear stress induces the translocation of Cx43 to the membrane surface and that unapposed hemichannels formed by Cx43 serve as a novel portal for the release of PGE2 in response to mechanical strain.     

Hemichannels formed by connexin 43 play an important role in the release of prostaglandin E(2) by osteocytes in response to mechanical strain

Osteocytes embedded in the matrix of bone are mechanosensory cells that translate strain into signals and regulate bone remodeling. Our previous studies using osteocyte-like MLO-Y4 cells have shown that fluid flow shear stress (FFSS) increases connexin (Cx) 43 protein expression, prostaglandin E(2) (PGE(2)) release, and intercellular coupling, and PGE(2) is an essential mediator between FFSS and gap junctions. However, the role of Cx43 in the release of PGE(2) in response to FFSS is unknown. Here, the FFSS-loaded MLO-Y4 cells with no or few intercellular channels released significantly more PGE(2) per cell than those cells at higher densities. Antisense Cx43 oligonucleotides and 18 beta-glycyrrhetinic acid, a specific gap junction and hemichannel blocker, significantly reduced PGE(2) release by FFSS at all cell densities tested, especially cells at the lowest density without gap junctions. FFSS, fluid flow-conditioned medium, and PGE(2) increased the activity of dye uptake. Moreover, FFSS induced Cx43 to migrate to the surface of the cell; this surface expressed Cx43 developed resistance to Triton-X-100 solublization. Our results suggest that hemichannels formed by Cx43, instead of intercellular channels, are likely to play a predominant role in the release of intracellular PGE(2) in response to FFSS.     

Osteoblasts and osteocytes respond differently to oscillatory and unidirectional fluid flow profiles

Bone cells subjected to mechanical loading by fluid shear stress undergo significant architectural and biochemical changes. The models of shear stress used to analyze the effects of loading bone cells in vitro include both oscillatory and unidirectional fluid shear profiles. Although the fluid flow profile experienced by cells within bone is most likely oscillatory in nature, to date there have been few direct comparisons of how bone cells respond to these two fluid flow profiles. In this study we evaluated morphologic and biochemical responses to a time course of unidirectional and oscillatory fluid flow in two commonly used bone cell lines, MC3T3-E1 osteoblasts and MLO-Y4 osteocytes. We determined that stress fibers formed and aligned within osteoblasts after 1 h of unidirectional fluid flow, but this response was not observed until greater than 5 h of oscillatory fluid flow. Despite the delay in stress fiber formation, oscillatory and unidirectional fluid flow profiles elicited similar temporal effects on the induction of both cyclooxygenase-2 (Cox-2) and osteopontin protein expression in osteoblasts. Interestingly, MLO-Y4 osteocytes formed organized stress fibers after exposure to 24 h of unidirectional shear stress, while the number of dendritic processes per cell increased along with Cox-2 protein levels after 24 h of oscillatory shear stress. Despite these differences, both flow profiles significantly altered osteopontin levels in MLO-Y4 osteocytes. Together these results demonstrate that the profile of fluid shear can induce significantly different responses from osteoblasts and osteocytes.     

Hemichannels Formed by Connexin 43 Play an Important Role in the Release of Prostaglandin E2 by Osteocytes in Response to Mechanical Strain

Osteocytes embedded in the matrix of bone are mechanosensory cells that translate strain into signals and regulate bone remodeling. Our previous studies using osteocyte-like MLO-Y4 cells have shown that fluid flow shear stress (FFSS) increases connexin (Cx) 43 protein expression, prostaglandin E₂ (PGE₂) release, and intercellular coupling, and PGE₂ is an essential mediator between FFSS and gap junctions. However, the role of Cx43 in the release of PGE₂ in response to FFSS is unknown. Here, the FFSS-loaded MLO-Y4 cells with no or few intercellular channels released significantly more PGE₂ per cell than those cells at higher densities. Antisense Cx43 oligonucleotides and 18 β-glycyrrhetinic acid, a specific gap junction and hemichannel blocker, significantly reduced PGE₂ release by FFSS at all cell densities tested, especially cells at the lowest density without gap junctions. FFSS, fluid flow-conditioned medium, and PGE₂ increased the activity of dye uptake. Moreover, FFSS induced Cx43 to migrate to the surface of the cell; this surface expressed Cx43 developed resistance to Triton-X-100 solublization. Our results suggest that hemichannels formed by Cx43, instead of intercellular channels, are likely to play a predominant role in the release of intracellular PGE₂ in response to FFSS.     

Expression of muscle anabolic and metabolic factors in mechanically loaded MLO-Y4 osteocytes

Lack of physical activity results in muscle atrophy and bone loss, which can be counteracted by mechanical loading. Similar molecular signaling pathways are involved in the adaptation of muscle and bone mass to mechanical loading. Whether anabolic and metabolic factors regulating muscle mass, i.e., insulin-like growth factor-I isoforms (IGF-I Ea), mechano growth factor (MGF), myostatin, vascular endothelial growth factor (VEGF), or hepatocyte growth factor (HGF), are also produced by osteocytes in bone in response to mechanical loading is largely unknown. Therefore, we investigated whether mechanical loading by pulsating fluid flow (PFF) modulates the mRNA and/or protein levels of muscle anabolic and metabolic factors in MLO-Y4 osteocytes. Unloaded MLO-Y4 osteocytes expressed mRNA of VEGF, HGF, IGF-I Ea, and MGF, but not myostatin. PFF increased mRNA levels of IGF-I Ea (2.1-fold) and MGF (2.0-fold) at a peak shear stress rate of 44Pa/s, but not at 22Pa/s. PFF at 22 Pa/s increased VEGF mRNA levels (1.8- to 2.5-fold) and VEGF protein release (2.0- to 2.9-fold). Inhibition of nitric oxide production decreased (2.0-fold) PFF-induced VEGF protein release. PFF at 22 Pa/s decreased HGF mRNA levels (1.5-fold) but increased HGF protein release (2.3-fold). PFF-induced HGF protein release was nitric oxide dependent. Our data show that mechanically loaded MLO-Y4 osteocytes differentially express anabolic and metabolic factors involved in the adaptive response of muscle to mechanical loading (i.e., IGF-I Ea, MGF, VEGF, and HGF). Similarly to muscle fibers, mechanical loading enhanced expression levels of these growth factors in MLO-Y4 osteocytes. Although in MLO-Y4 osteocytes expression levels of IGF-I Ea and MGF of myostatin were very low or absent, it is known that the activity of osteoblasts and osteoclasts is strongly affected by them. The abundant expression levels of these factors in muscle cells, in combination with low expression in MLO-Y4 osteocytes, provide a possibility that growth factors expressed in muscle could affect signaling in bone cells.     

Hemichannels Formed by Connexin 43 Play an Important Role in the Release of Prostaglandin E2by Osteocytes in Response to Mechanical Strain

Osteocytes embedded in the matrix of bone are mechanosensory cells that translate strain into signals and regulate bone remodeling. Our previous studies using osteocyte-like MLO-Y4 cells have shown that fluid flow shear stress (FFSS) increases connexin (Cx) 43 protein expression, prostaglandin E₂(PGE₂) release, and intercellular coupling, and PGE₂is an essential mediator between FFSS and gap junctions. However, the role of Cx43 in the release of PGE₂in response to FFSS is unknown. Here, the FFSS-loaded MLO-Y4 cells with no or few intercellular channels released significantly more PGE₂per cell than those cells at higher densities. Antisense Cx43 oligonucleotides and 18 β-glycyrrhetinic acid, a specific gap junction and hemichannel blocker, significantly reduced PGE₂release by FFSS at all cell densities tested, especially cells at the lowest density without gap junctions. FFSS, fluid flow-conditioned medium, and PGE₂increased the activity of dye uptake. Moreover, FFSS induced Cx43 to migrate to the surface of the cell; this surface expressed Cx43 developed resistance to Triton-X-100 solublization. Our results suggest that hemichannels formed by Cx43, instead of intercellular channels, are likely to play a predominant role in the release of intracellular PGE₂in response to FFSS.     

The P2X7 nucleotide receptor mediates skeletal mechanotransduction

The P2X7 nucleotide receptor (P2X7R) is an ATP-gated ion channel expressed in many cell types including osteoblasts and osteocytes. Mice with a null mutation of P2X7R have osteopenia in load bearing bones, suggesting that the P2X7R may be involved in the skeletal response to mechanical loading. We found the skeletal sensitivity to mechanical loading was reduced by up to 73% in P2X7R null (knock-out (KO)) mice. Release of ATP in the primary calvarial osteoblasts occurred within 1 min of onset of fluid shear stress (FSS). After 30 min of FSS, P2X7R-mediated pore formation was observed in wild type (WT) cells but not in KO cells. FSS increased prostaglandin (PG) E2 release in WT cells but did not alter PGE2 release in KO cells. Studies using MC3T3-E1 osteoblasts and MLO-Y4 osteocytes confirmed that PGE2 release was suppressed by P2X7R blockade, whereas the P2X7R agonist BzATP enhanced PGE2 release. We conclude that ATP signaling through P2X7R is necessary for mechanically induced release of prostaglandins by bone cells and subsequent osteogenesis.     

Early responses to mechanical load in tendon: role for calcium signaling, gap junctions and intercellular communication

Tendon and other connective tissue cells are subjected to diverse mechanical loads during daily activities. Thus, fluid flow, strain, shear and combinations of these stimuli activate mechanotransduction pathways that modulate tissue maintenance, repair and pathology. Early mechanotransduction events include calcium (Ca2+) signaling and intercellular communication. These responses are mediated through multiple mechanisms involving stretch-activated channels, voltage-activated channels such as Ca(v)1, purinoceptors, adrenoceptors, ryanodine receptor-mediated Ca2+ release, gap junctions and connexin hemichannels. Calcium, diacylglycerol, inositol (1,4,5)-trisphosphate, nucleotides and nucleosides play intracellular and/or extracellular signaling roles in these pathways. In addition, responses to mechanical loads in tendon cells vary among species, tendon type, anatomic location, loading conditions and other factors. This review includes a synopsis of the immediate responses to mechanical loading in connective tissue cells, particularly tenocytes. These responses involve Ca2+ signaling, gap junctions and intercellular communication.     

Correlation of cell strain in single osteocytes with intracellular calcium,but not intracellular nitric oxide,in response to fluid flow

Osteocytes compose 90–95% of all bone cells and are the mechanosensors of bone. In this study, the strain experienced by individual osteocytes resulting from an applied fluid flow shear stress was quantified and correlated to two biological responses measured in real-time within the same individual osteocytes: (1) the upregulation of intracellular calcium and (2) changes in intracellular nitric oxide. Osteocyte-like MLO-Y4 cells were loaded with Fluo-4 AM and DAR-4M and exposed to uniform laminar fluid flow shear stresses of 2, 8, or 16 dyn/cm². Intracellular calcium and nitric oxide changes were determined by measuring the difference in fluorescence intensity from the cell’s basal level prior to fluid flow and the level immediately following exposure. Individual cell strains were calculated using digital image correlation. MLO-Y4 cells showed a linear increase in cell strain, intracellular calcium concentration, and nitric oxide concentration with an increase in applied fluid flow rate. The increase in intracellular calcium was well correlated to the strain that each cell experienced. This study shows that osteocytes exposed to the same fluid flow experienced a range of individual strains and changes in intracellular calcium and nitric oxide concentrations, and the changes in intracellular calcium were correlated with cell strain. These results are among the first to establish a relationship between the strain experienced by osteocytes in response to fluid flow shear and a biological response at the single cell level. Mechanosensing and chemical signaling in osteocytes has been hypothesized to occur at the single cell level, making it imperative to understand the biological response of the individual cell.     

Activation of the Cpx phosphorelay signal transduction system in acidic phospholipid-deficient pgsA mutant cells of Escherichia coli

Low-intensity pulsed ultrasound (LIPUS) has been used as a safe and effective modality to enhance fracture healing. As the most abundant cells in bone, osteocytes orchestrate biological activities of effector cells via direct cell-to-cell contacts and by soluble factors. In this study, we have used the osteocytic MLO-Y4 cells to study the effects of conditioned medium from LIPUS-stimulated MLO-Y4 cells on proliferation and differentiation of osteoblastic MC3T3-E1 cells. Conditioned media from LIPUS-stimulated MLO-Y4 cells (LIPUS-Osteocyte-CM) were collected and added on MC3T3-E1 cell cultures. MC3T3-E1 cells cultured in LIPUS-Osteocyte-CM demonstrated a significant inhibition of proliferation and an increased alkaline phosphatase activity. The results of PGE(2) and NO assay showed that LIPUS could enhance PGE(2) and NO secretion from MLO-Y4 cells at all time points within 24h after LIPUS stimulation. We conclude that LIPUS regulates proliferation and differentiation of osteoblasts through osteocytes in vitro. Increased secretion of PGE(2) from osteocytes may play a role in this effect.     

Adaptation of connexin 43-hemichannel prostaglandin release to mechanical loading

Bone tissues respond to mechanical loading/unloading regimens to accommodate (re)modeling requirements; however, the underlying molecular mechanism responsible for these responses is largely unknown. Previously, we reported that connexin (Cx) 43 hemichannels in mechanosensing osteocytes mediate the release of prostaglandin, PGE(2), a crucial factor for bone formation in response to anabolic loading. We show here that the opening of hemichannels and release of PGE(2) by shear stress were significantly inhibited by a potent antibody we developed that specifically blocks Cx43-hemichannels, but not gap junctions or other channels. The opening of hemichannels and release of PGE(2) are magnitude-dependent on the level of shear stress. Insertion of a rest period between stress enhances this response. Hemichannels gradually close after 24 h of continuous shear stress corresponding with reduced Cx43 expression on the cell surface, thereby reducing any potential negative effects of channels staying open for extended periods. These data suggest that Cx43-hemichannel activity associated with PGE(2) release is adaptively regulated by mechanical loading to provide an effective means of regulating levels of extracellular signaling molecules responsible for initiation of bone (re)modeling.     

E11/gp38 selective expression in osteocytes: regulation by mechanical strain and role in dendrite elongation

Within mineralized bone, osteocytes form dendritic processes that travel through canaliculi to make contact with other osteocytes and cells on the bone surface. This three-dimensional syncytium is thought to be necessary to maintain viability, cell-to-cell communication, and mechanosensation. E11/gp38 is the earliest osteocyte-selective protein to be expressed as the osteoblast differentiates into an osteoid cell or osteocyte, first appearing on the forming dendritic processes of these cells. Bone extracts contain large amounts of E11, but immunostaining only shows its presence in early osteocytes compared to more deeply embedded cells, suggesting epitope masking by mineral. Freshly isolated primary osteoblasts are negative for E11 expression but begin to express this protein in culture, and expression increases with time, suggesting differentiation into the osteocyte phenotype. Osteoblast-like cell lines 2T3 and Oct-1 also show increased expression of E11 with differentiation and mineralization. E11 is highly expressed in MLO-Y4 osteocyte-like cells compared to osteoblast cell lines and primary osteoblasts. Differentiated, mineralized 2T3 cells and MLO-Y4 cells subjected to fluid flow shear stress show an increase in mRNA for E11. MLO-Y4 cells show an increase in dendricity and elongation of dendrites in response to shear stress that is blocked by small interfering RNA specific to E11. In vivo, E11 expression is also increased by a mechanical load, not only in osteocytes near the bone surface but also in osteocytes more deeply embedded in bone. Maximal expression is observed not in regions of maximal strain but in a region of potential bone remodeling, suggesting that dendrite elongation may be occurring during this process. These data suggest that osteocytes may be able to extend their cellular processes after embedment in mineralized matrix and have implications for osteocytic modification of their microenvironment.     

Connexin 32 and 43 gap junctions differentially modulate tenocyte response to cyclic mechanical load

Gap junctions allow rapid exchange of ions and small metabolites between cells. They can occur between connective tissue cells, and in tendons there are two prominent types, composed of connexin 32 or 43. These form distinct networks - tenocyte rows are linked by both longitudinally, but only by connexin 43 laterally. We hypothesised that the junctions had different roles in cell response to mechanical loading, and measured the effects of inhibitors of gap junction function on secretion of collagen by tenocyte cultures exposed to mechanical strain. Chicken tendon fibroblasts were exposed to cyclic tensile loading in the presence or absence of general gap junction inhibitors (halothane or the biomimetic peptide gap27), or antisense oligonucleotides to chicken connexin 32 or 43. Untreated cultures increased collagen secretion by around 25% under load. Halothane eliminated this response but caused cell damage. Gap27 peptide reduced secretion but maintained loading effects - strained cultures secreting more collagen than unstrained. Antisense downregulation showed major differences between connexins: antisense 32 reduced, and antisense 43 increased, collagen secretion. In both cases loading effects were maintained. This shows that (i) gap junctional integration of signals is important in load response of tenocyte populations - mechanotransduction occurs in individual cells but integration of signals markedly enhances it and (ii) communication via connexin 32 and 43 have differential effects on the load response, with connexin 32 being stimulatory and connexin 43 being inhibitory. Cells coordinate and control their response to mechanical signals at least in part by differential actions of these two types of gap junction.     

MT1-MMP modulates the mechanosensitivity of osteocytes

Membrane-type matrix metalloproteinase-1 (MT1-MMP) is expressed by mechanosensitive osteocytes and affects bone mass. The extracellular domain of MT1-MMP is connected to extracellular matrix, while its intracellular domain is a strong modulator of cell signaling. In theory MT1-MMP could thus transduce mechanical stimuli into a chemical response. We hypothesized that MT1-MMP plays a role in the osteocyte response to mechanical stimuli. MT1-MMP-positive and knockdown (siRNA) MLO-Y4 osteocytes were mechanically stimulated with a pulsating fluid flow (PFF). Focal adhesions were visualized by paxillin immunostaining. Osteocyte number, number of empty lacunae, and osteocyte morphology were measured in long bones of MT1-MMP(+/+) and MT1-MMP(-/-) mice. PFF decreased MT1-MMP mRNA and protein expression in MLO-Y4 osteocytes, suggesting that mechanical loading may affect pericellular matrix remodeling by osteocytes. MT1-MMP knockdown enhanced NO production and c-jun and c-fos mRNA expression in response to PFF, concomitantly with an increased number and size of focal adhesions, indicating that MT1-MMP knockdown osteocytes have an increased sensitivity to mechanical loading. Osteocytes in MT1-MMP(-/-) bone were more elongated and followed the principle loading direction, suggesting that they might sense mechanical loading. This was supported by a lower number of empty lacunae in MT1-MMP(-/-) bone, as osteocytes lacking mechanical stimuli tend to undergo apoptosis. In conclusion, mechanical stimulation decreased MT1-MMP expression by MLO-Y4 osteocytes, and MT1-MMP knockdown increased the osteocyte response to mechanical stimulation, demonstrating a novel and unexpected role for MT1-MMP in mechanosensing.     

The effect of cytoskeletal disruption on pulsatile fluid flow-induced nitric oxide and prostaglandin E2 release in osteocytes and osteoblasts

Fluid flowing through the bone porosity might be a primary stimulus for functional adaptation of bone. Osteoblasts, and osteocytes in particular, respond to fluid flow in vitro with enhanced nitric oxide (NO) and prostaglandin E(2) (PGE(2)) release; both of these signaling molecules mediate mechanically-induced bone formation. Because the cell cytoskeleton is involved in signal transduction, we hypothesized that the pulsatile fluid flow-induced release of NO and PGE(2) in both osteoblastic and osteocytic cells involves the actin and microtubule cytoskeleton. In testing this hypothesis we found that fluid flow-induced NO response in osteoblasts was accompanied by parallel alignment of stress fibers, whereas PGE(2) response was related to fluid flow stimulation of focal adhesions formed after cytoskeletal disruption. Fluid flow-induced PGE(2) response in osteocytes was inhibited by cytoskeletal disruption, whereas in osteoblasts it was enhanced. These opposite PGE(2) responses are likely related to differences in cytoskeletal composition (osteocyte structure was more dependent on actin), but may occur via cytoskeletal modulation of shear/stretch-sensitive ion channels that are known to be dominant in osteocyte (and not osteoblast) response to mechanical loading.     

A role for the primary cilium in paracrine signaling between mechanically stimulated osteocytes and mesenchymal stem cells

Bone turnover is a mechanically regulated process, coordinated in part by the network of mechanosensitive osteocytes residing within the tissue. The recruitment and bone forming activity of the mesenchymal derived osteoblast is determined by numerous factors including mechanical loading. It is therefore somewhat surprising that although mechanically regulated signaling between the coordinating osteocytes and mesenchymal stem cells (MSCs) should exist, to date it has not been directly demonstrated. In this study, conditioned media from mechanically stimulated osteocytes (MLO-Y4 cell line) was collected and added to MSCs (C3H10T1/2 cell line). The addition of mechanically stimulated osteocyte conditioned media resulted in a significant upregulation of the osteogenic genes OPN and COX-2 in MSCs compared to statically cultured conditioned media, demonstrating a novel paracrine signaling mechanism between the two cell types. The same mechanically conditioned media did not alter gene expression in osteoblasts (MC3T3 cell line), and mechanically stimulated osteoblast conditioned media did not alter gene expression in MSCs demonstrating that this signaling is unique to osteocytes and MSCs. Finally, the upregulation in osteogenic genes in MSCs was not observed if primary cilia formation was inhibited prior to mechanical stimulation of the osteocyte. In summary, the results of this study indicate that soluble factors secreted by osteocytes in response to mechanical stimulation can enhance osteogenic gene expression in MSCs demonstrating a novel, unique signaling mechanism and introduces a role for the primary cilium in flow mediated paracrine signaling in bone thereby highlighting the cilium as a potential target for therapeutics aimed at enhancing bone formation.