Astrid D. Bakker

The production of nitric oxide and prostaglandin E2 by primary bone cells is shear stress dependent

Loading-induced flow of interstitial fluid through the lacuno-canalicular network is a likely signal for bone cell adaptive responses. However, the nature of the stimulus that activates the cell is debated. Candidate stimuli include wall shear stress, streaming potentials, and chemotransport. We have addressed the nature of the flow-derived cell stimulus by comparing variations in fluid transport with variations in wall shear stress, using nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production as a parameter of bone cell activation. Adult mouse long bone cell cultures were treated for 15min with or without pulsating fluid flow using the following regimes: Low PFF, mean flow rate 0.20 cm(3)/s, 3 Hz, shear stress 0.4+/-0.12 Pa; Medium PFF, 0.33 cm(3)/s, 5 Hz, 0.6+/-0.27 Pa; and High PFF, 0.63 cm(3)/s, 9Hz, 1.2+/-0.37 Pa. In some Low PFF experiments, 2.8% neutral dextran (mol. wt. 4.98x10(4)) was added to the flow medium to increase the viscosity, thereby increasing the wall shear stress 3-fold to a level similar of the High PFF stimulus, but without affecting streaming potentials or chemotransport. NO and PGE(2) production were stimulated by Low, Medium, and High PFF in a dose-dependent manner. Application of Low PFF using dextran-supplemented medium, enhanced both the NO and PGE(2) response by 3-fold, to a level mimicking the response to High PFF at normal viscosity. These results show that the production of NO and PGE(2) by bone cells can be enhanced in a dose-dependent manner by fluid flow of increasing wall shear stress. Therefore, the stimulus leading to NO and PGE(2) production is the flow-derived shear stress, and not streaming potentials or chemotransport.

Mechanosensation and transduction in osteocytes

The human skeleton is a miracle of engineering, combining both toughness and light weight. It does so because bones possess cellular mechanisms wherein external mechanical loads are sensed. These mechanical loads are transformed into biological signals, which ultimately direct bone formation and/or bone resorption. Osteocytes, since they are ubiquitous in the mineralized matrix, are the cells that sense mechanical loads and transduce the mechanical signals into a chemical response. The osteocytes then release signaling molecules, which orchestrate the recruitment and activity of osteoblasts or osteoclasts, resulting in the adaptation of bone mass and structure. In this review, we highlight current insights in bone adaptation to external mechanical loading, with an emphasis on how a mechanical load placed on whole bones is translated and amplified into a mechanical signal that is subsequently sensed by the osteocytes.

PLS3 Mutations in X-Linked Osteoporosis with Fractures

Plastin 3 (PLS3), a protein involved in the formation of filamentous actin (F-actin) bundles, appears to be important in human bone health, on the basis of pathogenic variants in PLS3 in five families with X-linked osteoporosis and osteoporotic fractures that we report here. The bone-regulatory properties of PLS3 were supported by in vivo analyses in zebrafish. Furthermore, in an additional five families (described in less detail) referred for diagnosis or ruling out of osteogenesis imperfecta type I, a rare variant (rs140121121) in PLS3 was found. This variant was also associated with a risk of fracture among elderly heterozygous women that was two times as high as that among noncarriers, which indicates that genetic variation in PLS3 is a novel etiologic factor involved in common, multi-factorial osteoporosis.

Pulsating fluid flow modulates gene expression of proteins involved in Wnt signaling pathways in osteocytes

Strain‐derived flow of interstitial fluid activates signal transduction pathways in osteocytes that regulate bone mechanical adaptation. Wnts are involved in this process, but whether mechanical loading modulates Wnt signaling in osteocytes is unclear. We assessed whether mechanical stimulation by pulsating fluid flow (PFF) leads to functional Wnt production, and whether nitric oxide (NO) is important for activation of the canonical Wnt signaling pathway in MLO‐Y4 osteocytes. MC3T3‐E1 osteoblasts were studied as a positive control for the MLO‐Y4 osteocyte response to mechanical loading. MLO‐Y4 osteocytes and MC3T3‐E1 osteoblasts were submitted to 1‐h PFF (0.7 ± 0.3 Pa, 5 Hz), and postincubated (PI) without PFF for 0.5–3 h. Gene expression of proteins related to the Wnt canonical and noncanonical pathways were studied using real‐time polymerase chain reaction (PCR). In MLO‐Y4 osteocytes, PFF upregulated gene expression of Wnt3a, c‐jun, connexin 43, and CD44 at 1–3‐h PI. In MC3T3‐E1 osteoblasts, PFF downregulated gene expression of Wnt5a and c‐jun at 0.5–3‐h PI. In MLO‐Y4 osteocytes, gene expression of PFF‐induced Wnt target genes was suppressed by the Wnt antagonist sFRP4, suggesting that loading activates the Wnt canonical pathway through functional Wnt production. The NO inhibitor L‐NAME suppressed the effect of PFF on gene expression of Wnt target genes, suggesting that NO might play a role in PFF‐induced Wnt production. The response to PFF differed in MC3T3‐E1 osteoblasts. Because Wnt signaling is important for bone mass regulation, osteocytes might orchestrate loading‐induced bone remodeling through, among others, Wnts.

Early activation of the beta-catenin pathway in osteocytes is mediated by nitric oxide, phosphatidyl inositol-3 kinase/Akt, and focal adhesion kinase

Bone mechanotransduction is vital for skeletal integrity. Osteocytes are thought to be the cellular structures that sense physical forces and transform these signals into a biological response. The Wnt/beta-catenin signaling pathway has been identified as one of the signaling pathways that is activated in response to mechanical loading, but the molecular events that lead to an activation of this pathway in osteocytes are not well understood. We assessed whether nitric oxide, focal adhesion kinase, and/or the phosphatidyl inositol-3 kinase/Akt signaling pathway mediate loading-induced beta-catenin pathway activation in MLO-Y4 osteocytes. We found that mechanical stimulation by pulsating fluid flow (PFF, 0.7+/-0.3 Pa, 5 Hz) for 30 min induced beta-catenin stabilization and activation of the Wnt/beta-catenin signaling pathway. The PFF-induced stabilization of beta-catenin and activation of the beta-catenin signaling pathway was abolished by adding focal kinase inhibitor FAK inhibitor-14 (50 microM), or phosphatidyl inositol-3 kinase inhibitor LY-294002 (50 microM). Addition of nitric oxide synthase inhibitor L-NAME (1.0mM) also abolished PFF-induced stabilization of beta-catenin. This suggests that mechanical loading activates the beta-catenin signaling pathway by a mechanism involving nitric oxide, focal adhesion kinase, and the Akt signaling pathway. These data provide a framework for understanding the role of beta-catenin in mechanical adaptation of bone.

Inhibition of osteocyte apoptosis by fluid flow is mediated by nitric oxide

Bone unloading results in osteocyte apoptosis, which attracts osteoclasts leading to bone loss. Loading of bone drives fluid flow over osteocytes which respond by releasing signaling molecules, like nitric oxide (NO), that inhibit osteocyte apoptosis and alter osteoblast and osteoclast activity thereby preventing bone loss. However, which apoptosis-related genes are modulated by loading is unknown. We studied apoptosis-related gene expression in response to pulsating fluid flow (PFF) in osteocytes, osteoblasts, and fibroblasts, and whether this is mediated by loading-induced NO production. PFF (0.7+/-0.3Pa, 5Hz, 1h) upregulated Bcl-2 and downregulated caspase-3 expression in osteocytes. l-NAME attenuated this effect. In osteocytes PFF did not affect p53 and c-Jun, but l-NAME upregulated c-Jun expression. In osteoblasts and fibroblasts PFF upregulated c-Jun, but not Bcl-2, caspase-3, and p53 expression. This suggests that PFF inhibits osteocyte apoptosis via alterations in Bcl-2 and caspase-3 gene expression, which is at least partially regulated by NO.

Mechanotransduction in bone cells proceeds via activation of COX-2, but not COX-1

Cyclooxygenase (COX) is the key enzyme in the production of prostaglandins, which are essential for the response of bone to mechanical loading. We determined which COX-isoform, COX-1 or COX-2, determines loading-induced prostaglandin production in primary bone cells in vitro. Mouse and human bone cells reacted to 1 h of pulsating fluid flow (PFF, 0.6+/-0.3 Pa at 5 Hz) with an increased prostaglandin E(2) production, which continued 24 h after cessation of PFF. Inhibition of COX-2 activity with NS-398 abolished the stimulating effect of PFF both at 1 h and at 24 h post-incubation, while inhibition of COX-1 by SC-560 affected neither the early nor the late response to flow. PFF rapidly stimulated COX-2 mRNA expression at 1 h but did not affect COX-1 mRNA expression. COX-2 mRNA expression was still significantly enhanced 24 h after cessation of PFF. We conclude that COX-2 is the mechanosensitive form of COX that determines the response of bone tissue to mechanical loading.

The role of osteocytes in bone mechanotransduction

IntroductionBones are subjected to a variety of mechanical loads duringdaily activities. In the nineteenth century, Julius Wolffproposed that bones adapt their mass and 3D structure tothe loading conditions in order to optimize their load-bearing capacity, and that this process is driven bymechanical stress [1]. For the past centuries, an increasingnumber of theoretical and experimental results reveal thatosteocytes are the pivotal cells orchestrating this bio-mechanical regulation of bone mass and structure, whichis accomplished by the process of bone remodeling [2–5]Osteocytes are terminally differentiated cells of theosteogenic lineage that are derived from mesenchymalprecursor cells. A number of molecules have been iden-tified as important markers of osteocytes, such as matrixextracellular phosphoglycoprotein [6] sclerostin [7], dentinmatrix protein-1 [8], and phex protein [8]. The osteocytesare the most abundant cells in adult bone and are constantlyspaced throughout the mineralized matrix. Mature osteo-cytes have a characteristic dendritic cell shape, with pro-cesses radiating from the cell body through the canaliculi indifferent directions. These processes form an intercellularnetwork through gap and adherent junctions with surround-ing osteocytes, the cells lining the bone surface and bonemarrow. Through this unique 3D network, osteocytes areanatomically placed in a prime position not only to sensedeformations driven by stresses placed upon bone, butalso to respond with passage of signals to the neighboringcells [9].For more than a decade now, it is known that theosteocytes are very sensitive to stress applied to intact bonetissue [10–16]. Computer simulation models have shownthat mechanosensors lying at the surface of bone, asosteoblasts and bone lining cells do, would be less sensitiveto changes in the loading pattern than the osteocytes, lyingwithin the calcified matrix [3]. Interestingly, targetedablation of osteocytes in mice disturbs the adaptation ofbone to mechanical loading [16].Osteocytes as key players in the process of bonemechanotransductionIt is currently believed that when bones are loaded, theresulting deformation will drive the thin layer of interstitialfluid surrounding the network of osteocytes to flow fromregions under high pressure to regions under low pressure[17, 18]. This flow of fluid is sensed by the osteocyteswhich in turn produce signaling molecules that can regulatebone resorption through the osteoclasts, and bone formationthrough the osteoblasts, leading to adequate bone remodel-ing [17, 18]. This concept is known as the fluid flowhypothesis. Evidence has been increasing for the flow ofcanalicular interstitial fluid as the likely factor that informsthe osteocytes about the level of bone loading [2, 5, 17, 18].Nevertheless, Vatsa and colleagues [19, 20] proposed thatif osteocytes could sense matrix strains directly, the cellshape, cytoskeletal alignment and distribution of adhesionsites in osteocytes in situ would bear alignment to themechanical loading patterns. Indeed, it was shown that the

Androgen receptor disruption increases the osteogenic response to mechanical loading in male mice

In female mice, estrogen receptor‐alpha (ERα) mediates the anabolic response of bone to mechanical loading. Whether ERα plays a similar role in the male skeleton and to what extent androgens and androgen receptor (AR) affect this response in males remain unaddressed. Therefore, we studied the adaptive response of in vivo ulna loading in AR‐ERα knockout (KO) mice and corresponding male and female single KO and wild‐type (WT) littermates using dynamic histomorphometry and immunohistochemistry. Additionally, cultured bone cells from WT and AR KO mice were subjected to mechanical loading by pulsating fluid flow in the presence or absence of testosterone. In contrast with female mice, ERα inactivation in male mice had no effect on the response to loading. Interestingly, loading induced significantly more periosteal bone formation in AR KO (+320%) and AR‐ERα KO mice (+256%) compared with male WT mice (+114%) and had a stronger inhibitory effect on SOST/sclerostin expression in AR KO versus WT mice. In accordance, the fluid flow‐induced nitric oxide production was higher in the absence of testosterone in bone cells from WT but not AR KO mice. In conclusion, AR but not ERα activation limits the osteogenic response to loading in male mice possibly via an effect on WNT signaling.

WNT5A induces osteogenic differentiation of human adipose stem cells via rho-associated kinase Rock

BACKGROUND AIMS Human (h) adipose tissue-derived mesenchymal stromal cells (ASC) constitute an interesting cellular source for bone tissue engineering applications. Wnts, for example Wnt5a, are probably important regulators of osteogenic differentiation of stem cells, but the role of Wnt5a in hASC lineage commitment and the mechanisms activated upon Wnt5a binding are unknown. We examined whether Wnt5a induces osteogenic and/or adipogenic differentiation of hASC. METHODS hASC were incubated for 7 days with or without Wnt5a, rho-associated kinase (ROCK)-activity inhibitor Y27632 or Wnt3a. Cells were lysed for total RNA isolation, DNA content and alkaline phosphatase (ALP) activity. Mineralized nodule formation and gene expression of osteogenic markers osteocalcin and runt-related protein-2 (RUNX2), and adipogenic markers peroxisome proliferator activator receptor-γ (PPARγ) and transcription factor apetala-2 (aP2), were analyzed. hASC were incubated with Wnt5a or Wnt3a to determine activation of canonical and/or non-canonical Wnt signaling pathways, and protein kinase C activity (PKC), total ß-catenin content and gene expression of connexin 43 and cyclin D1 were quantified. RESULTS Wnt5a increased ALP activity and RUNX2 and osteocalcin gene expression, and down-regulated adipogenic markers through ROCK activity. Wnt5a also induced mineralized nodule formation. Wnt3a only enhanced RUNX2 and osteocalcin gene expression, and did not induce osteogenic differentiation. Wnt5a activated the non-canonical Wnt signaling pathway by increasing PKC activity, while Wnt3a mildly activated the Wnt canonical pathway by increasing total ß-catenin content and connexin 43 and cyclin D1 gene expression. CONCLUSIONS Our data illustrate the importance of Wnt5a as a stimulator of hASC osteogenic differentiation, and show that changes in actin cytoskeleton controlled by ROCK are determinants for Wnt5a-induced osteogenic differentiation of hASC.