The Wnt pathway: an important control mechanism in bone’s response to mechanical loading

Abstract

The conversion of mechanical energy into biochemical changes within living cells is process known as mechanotransduction. Bone is a quintessential tissue for studying the molecular mechanisms of mechanotransduction, as the skeleton’s mechanical competence is crucial for vertebrate movement. Bone cell mechanotransduction is facilitated by a number of cell biological pathways, one of the most prominent of which is the Wnt signaling cascade. The Wnt co-receptor Lrp5 has been identified as a crucial protein for mechanical signaling in bone, and modifiers of Lrp5 activity play important roles in mediating signaling efficiency through Lrp5, including sclerostin, Dkk1, and the co-receptor Lrp4. Mechanical regulation of sclerostin is mediated by certain members of the Hdac family. Other mechanisms that influence Wnt signaling—some of which are mechanoresponsive—are coming to light, including R-spondins and their role in organizing the Rnf43/Znrf3 and Lgr4/5/6 complex that liberates Lrp5. While the identity of the key Wnt proteins involved in bone cell mechanical signaling are elusive, the likely pool of key players is narrowing. Identification of Wnt-based molecular targets that can be modulated pharmacologically to make mechanical stimulation (e.g., exercise) more beneficial is an emerging approach to improving skeletal integrity and reducing fracture risk.

One of the most beneficial and cost-effective measures that can be taken to promote overall health is exercise. Exercise has clear and lasting positive effects on numerous organ systems, physiologic processes, and psychological functions, not the least of which is musculoskeletal health. Exercise is effective in promoting bone health because skeletal cells are mechanosensitive; that is, they sense and respond to mechanical perturbation induced by physical stimulation, and generate signals that ultimately result in a more mechanically competent skeleton. With sedentary behavior at an all-time high in the US,⁽¹⁾ it is crucially important for individuals to exercise in a manner that maximizes the osteogenic output to a given physical activity. We have looked at recovery periods and cellular saturation, in conjunction with the timing of mechanical loading sessions, as one mechanism to get more “bang for your buck” when it comes to loading.^(2-6) However, a complementary approach is to discover biomolecular shortcuts to make the same mechanical input more osteoanabolic. Accordingly, there is considerable research effort focused on identifying cellular mechanisms that normally restrain (or activate) the osteoanabolic effects of mechanical loading. Conceptually, that information can be used to design a therapeutic agent to target those factors during a vigorous exercise session, so that the same session is more osteogenic when the inhibitor/agonist is taken. The first step in this approach—that is, making loading more beneficial to the skeleton—is identifying a molecular target, or one of its upstream/downstream mediators, that can be modulated to make mechanical stimulation more beneficial. Numerous pathways and cellular cascades have been implicated in the process of bone cell mechanotransduction (reviewed in⁽⁷⁾), but one key pathway with potent effects in bone, that has gained a great deal of traction both inside and outside of mechanotransduction is the Wnt signaling pathway. Targeting Wnt during exercise has the potential to not only improve bone mass and geometry, but also reduce fat mass and adipogenesis, as canonical Wnt signaling plays a significant role in mesenchymal cell fate.⁽⁸⁾

Experimental and clinical identification of Wnt as a mechanosensitive pathway

Study of the Wnt signaling pathway originated in the early 1980s, when it was discovered that integration of the mouse mammary tumor virus (MMTV) into the int-1 locus (now known as Wnt1) caused unrestrained Wnt1 expression and mammary tumor formation in mice.⁽⁹⁾ Over the following two decades, subsequent identification of the cell surface receptors involved in Wnt signal reception, the proteins involved in intracellular signal transduction, and the extracellular mediators of signaling efficiency all contributed to defining a role for Wnt in neural tube development, limb patterning, planar cell polarity, axon guidance, and further understanding of tumor biology. However, Wnt signaling has more recently emerged as a key pathway involved in bone homeostasis in general, and in mechanotransduction in particular.⁽¹⁰⁾ The first clues of Wnt’s role in bone biology began ~20 years ago with several gene mapping studies, which sought to identify the genetic cause of Osteoporosis Pseudoglioma (OPPG), an autosomal recessive disease characterized by early-onset osteoporosis and blindness. Those studies revealed that loss-of-function mutation in the Wnt co-receptor LDL-Receptor-related Protein 5 (LRP5) caused OPPG.⁽¹¹⁾ Shortly thereafter, other reports revealed that certain N-terminally located missense mutations in LRP5 are associated with a high bone mass (HBM) phenotype in humans.^(12-15) Thus, loss- and gain- of function mutations in LRP5 appear to have substantial and opposing effects on bone mass and strength, ranging from severe osteoporosis, bone deformity, and increased susceptibility to fracture, to osteosclerosis and increased fracture resistance.

Following the discovery that the Wnt co-receptor Lrp5 plays a major role in bone metabolism, the signaling pathways in which Lrp5 participates became of particular interest for bone biologists looking for therapeutic targets. The most widely studied pathway associated with Lrp5 activation is the “canonical” Wnt pathway. Canonical Wnt signaling begins by binding of secreted Wnt proteins to Lrp5 (or Lrp6) and a seven-pass transmembrane co-receptor called Frizzled (Fzd). Activation of the Lrp/Fzd receptor complex at the cell surface by Wnt ultimately leads to the nuclear accumulation of β-catenin, which complexes with members of the Tcf/Lef transcription factor family to regulate gene transcription (Fig. 1). Between the cell membrane (where Wnt, Lrp5, and Fzd interact) and the nucleus (where β-catenin accumulates) is a complex regulatory process that involves a large number of cytosolic proteins including disheveled (Dsh), Axin, adenomatous polyposis coli gene product (Apc), glycogen synthase kinase 3β, (Gsk3β), and casein kinase 1 (Ck1), among others.⁽¹⁶⁾ Normally, when Wnt signaling is not active, the Axin protein complex is intact and serves to bring Ck1 and Gsk3β into close proximity to β-catenin, where they can sequentially phosphorylate β-catenin.⁽¹⁷⁾ Phosphorylated (inactive) β-catenin is quickly ubiquitinated and consequently targeted for proteosomal degradation by the 26S proteosome. ⁽¹⁷⁾ Wnt signaling causes the Axin/Gsk3β complex to dismantle, which allows β-catenin to avoid phosphorylation by Ck1 and Gsk3β. Activated (non-phosphorylated) β-catenin can then accumulate in the cytoplasm, and subsequently translocate to the nucleus where it partners with certain members of the High Mobility Group Box family of transcription factors (e.g., Tcf/Lef1) and upregulates transcription of Wnt-responsive genes.

Figure 1:

[Left] Lack of Wnt signaling permits stabilization of the Axin-Apc complex, which facilitates phosphorylation of β-catenin by Gsk3 and Ck1. Phosphorylated β-catenin is subsequently ubiquitin-tagged for proteosome degradation, and consequently does not accumulate in the cytoplasm in sufficient quantities to allow translocation to the nucleus. [Right] Activated Wnt signaling occurs through Wnt binding and complexing of Lrp5/6 with Frizzled. Formation of the trimeric receptor–ligand complex induces degredation of the Axin complex. Loss of the β-catenin degradation machinery allows β-catenin to remain stable (unphosphorylated) and accumulate in the cytoplasm, to the point where translocation to the nucleus occurs and gene transcription is initiated.

With the generation of mice modeled after patients with Lrp5 mutations, significant insight into the mechanisms and extent of Lrp5’s effects on bone metabolism began coming to light. Mice engineered with loss-of-function mutations in Lrp5 consistently exhibit low bone mass.^{(18) (19-21)} However, the weight-bearing portions of the skeleton (long bones) bear a greater deficit in bone mass than the axial, non weight-bearing portions (e.g. lumbar vertebrae and skull).⁽¹⁸⁾ This condition is similar to the disuse phenotype that seen among spinal cord injury patients, in whom vertebral bone density is normal but bone density in the limb bones (below the lesioned cord level, e.g., femur) is significantly reduced.⁽²²⁾ Those observations suggested that the Wnt pathway might be a major regulator of mechanical signal transduction in bone cells. To test this proposal, we challenged Lrp5 knockout with a skeletal loading protocol (ulnar loading), and found a nearly complete obliteration in responsiveness to mechanical stimulation.⁽¹⁸⁾ Another group came to the same conclusion using tibia loading on an independently engineered Lrp5^−/− mouse model.⁽²³⁾ If loss of Lrp5 severely diminishes mechanotransduction in bone tissue, what about gain of function mutations in the same receptor? Do Lrp5 HBM-causing mutations confer increased sensitivity to mechanical loading? We tested this idea by generating mice with two of the most commonly found clinical HBM-causing point mutations in LRP5—G171V and A214V—knocked into the endogenous Lrp5 locus. These mice were subjected to in vivo tibia loading, and both missense knockin models exhibited greater osteogenic response per unit mechanical strain.⁽²⁴⁾ A similar result (increased responsiveness) was reported using a transgenic approach to overexpress the G171V mutation specifically in bone cells.⁽²³⁾ While there are no published data on exercise effects in the handful of identified OPPG patients (LRP5-deficient) or LRP5-HBM patients, clinical data from healthy adults support the role of LRP5 signaling in regulating bone mechanotransduction. In a large human sample, several single nucleotide polymorphisms (SNPs) in LRP5, located in exons 10 and 18, significantly affected the relation between physical activity and bone mass accrual.⁽²⁵⁾

Beyond mouse and human studies, a large body of literature using in vitro models implicates the Wnt pathway in mechanotransduction. For example, stretched osteoblasts harvested from transgenic mice expressing a Tcf/β-catenin transcription reporter (TopGal mice) exhibited activation of canonical Wnt signaling (β-galactosidase expression) after stretching.⁽²⁶⁾ In addition, fluid shear stress stimulates the translocation of β-catenin to the nucleus in MC3T3-E1 cells, UMR cells, and in primary rat calvarial osteoblasts.^(27,28) Furthermore, this translocation event—the hallmark of Wnt signaling—was accompanied by Gsk3β phosphorylation, suggesting that canonical Wnt signaling is activated.⁽²⁷⁾ Collectively, these experiments suggest that Wnt/Lrp5/β-catenin signaling is an integral part of the mechanosensory apparatus in bone tissue.

Extracellular modulators of Wnt signaling are mechanosensitive

A logical line of inquiry has emerged from these studies: how does the mechanical environment alter Lrp5 signaling in normal bone tissue? For example, increased mechanical stimulation could enhance Wnt signaling by (1) increasing Wnt expression/release; (2) changing receptor expression (e.g., upregulation Lrp and Fzd, downregulation of Krm or Lrp4); or (3) decreasing expression/release of secreted Wnt signaling antagonists (e.g., Wif1, sFrp1, Dkk1, Sost), among others. Regarding the latter possibility, the canonical Wnt pathway is modulated by a number of secreted proteins that primarily serve as antagonists, but may occasionally function as agonists. These secreted proteins inhibit Wnt signaling by primarily binding to Wnt proteins (e.g., Wifs, sFrps) or to the Lrp5/6 receptors (Dkk1, Sost/sclerostin, Sostdc1/Wise); in either case the ligand–receptor interaction is compromised (Fig. 2).

Figure 2:

Secreted Wnt signaling inhibitors antagonize Wnt signaling by binding Wnts (e.g., sFrp, Wif) or binding the Wnt receptors Lrp5/6 (Dkk1, Sost/sclerostin, and Sostdc1/Wise). Dkk1 is known to induce endocytosis of the Lrp receptors after binding with Kremen. Lrp4 mediates sclerostin binding to Lrp5.

Sclerostin, the protein product of the Sost gene, is perhaps the most widely studied Lrp5/6 antagonist in the skeletal biology field. Sclerostin is an osteocyte-enriched cysteine-knot secreted glycoprotein that is a potent inhibitor of bone formation.⁽²⁹⁾ Mutations in the Sost gene, or in its distant regulatory elements, cause sclerosing bone disorders such as sclerosteosis and Van Buchem’s disease.^(30-32) Similar to the Lrp5 HBM patients, individuals with Sost mutations exhibit very high bone mass in the appendicular and axial skeleton.^(33-35) Mice engineered with loss-of-function mutations in the Sost gene exhibit very high bone mass,⁽³⁶⁾ while overexpression of Sost results in low bone mass.⁽³⁷⁾ Unlike Dkk1 (another potent Lrp5/6 inhibitor, see below), which binds to first and third β-propeller domain of Lrp5,⁽³⁸⁾ sclerostin binds exclusively to the first β-propeller of the Lrp5 receptor.⁽³⁹⁾ Interestingly, while both Dkk1 and sclerostin bind and inhibit Lrp5/6 (and do so at different motifs on the receptor) the presence of both inhibitors does not impair Lrp5/6 signaling more than either inhibitor expressed alone. Moreover, when present in equal amounts, Dkk1 and sclerostin do not simultaneously bind the same receptor. Dkk1 outcompetes sclerostin for binding to Lrp5/6, and can even displace pre-bound sclerostin from the receptor.⁽⁴⁰⁾ Until recently, it was assumed that sclerostin bound and inhibited Lrp5 without interaction from other molecules. However, another LDL-receptor related protein— Lrp4—facilitates sclerostin action on Lrp5/6, and ultimately, Wnt inhibition. This discovery is based on the identification of certain LRP4 missense mutations among humans that result in an HBM phenotype.^(41,42) We engineered an Lrp4 mutant mouse model based on one of the human families – an R1170W knockin. These mice exhibit high bone mass, are resistant to the osteopenic effects of Sost overexpression, and are resistant to the bone-wasting effects of mechanical disuse.⁽⁴³⁾ Thus, much like Krm proteins facilitate Dkk1 action on Lrp5, Lrp4 appears to facilitate sclerostin’s action on Lrp5.

Sclerostin expression in adult bone is largely limited to osteocytes,^(29,44) which have long been postulated as the “mechanosensor” cells in bone.^(45,46) The osteocyte population density, distribution, and extensive communication networks within bone make these cells ideal mechanosensors in bone’s adaptive process. Teleologically, sclerostin presents a particularly attractive candidate for regulating mechanically-induced signaling through the Lrp5 receptors. Because sclerostin is a potent inhibitor of Lrp5, and because Lrp5 is required for mechanotransduction, we investigated whether modulation of sclerostin levels might be a mechanism by which mechanical loading modulates Lrp5 activity during loading. We investigated the regulation of Sost/sclerostin by the mechanical environment using a murine model of enhanced loading (axial ulnar loading).⁽⁴⁷⁾ Ulnar loading in rodents induces a consistent pattern of bone deformation (strain) both along the bone axis and cross-sectionally at the midshaft. Sost/sclerostin levels were reduced dramatically by ulnar loading (Fig 3). Portions of the ulnar shaft receiving a greater strain stimulus were associated with a greater reduction in Sost/sclerostin than portions of the shaft enduring lower strains.

Figure 3:

Mechanical loading induced increases bone formation and decreased sclerostin staining, particularly at the high strain locations of the ulnar diaphysis.⁽⁴⁷⁾ The upper image depicts load-induced bone formation rates (rBFR/BS), which were least at the lowest strain location (proximal), greater at the moderately-strained midshaft, and greatest at high strain location (distal) as indicated by the proximo-distal gradient in fluorochrome labeling of the periosteal surfaces. The lower image depicts the load-induced decrease in the percent of osteocytes staining positive for sclerostin (r%Sclr+), which was least at the lowest strain location (proximal), greater (though not significantly) at the moderately-strained midshaft, and greatest at high strain location (distal). Thus bone formation rates, measured 1-2 weeks after loading, were greatest at diaphyseal locations exhibiting the greatest reduction in sclerostin, measured a few days after loading, and vice versa. n=7-9 mice (16-wk old female C57BL/6) per group. *=significantly different from the proximal site; †=significantly different from the midshaft site, at p<0.05.

While it is clear that mechanical stimulation modulates sclerostin levels, it remained to be established whether this phenomenon had functional consequences. To address this issue, we next investigated whether the load-induced reduction in sclerostin is required for bone mechanotransduction to occur. This was accomplished by engineering a transgenic mouse that expressed Sost in osteocytes, but expression was driven using a promoter that would not undergo downregulation in response to loading (^8kbDmp1-Sost). The ^8kbDmp1-Sost mouse maintains high levels of sclerostin after loading, and consequently, permitted evaluation of how efficiently mechanotransduction can occur if Sost levels are not permitted to decline. Load induced bone formation was significantly reduced in the transgenic mice, to a similar degree to that observed in Lrp5^−/− mice.⁽⁴⁸⁾ These data suggest that downregulation of Sost and/or reduction in sclerostin protein is required for mechanotransduction to occur, further supporting previous data on the mechanism of action for Lrp5 activity during mechanical stimulation. More broadly, these data suggest that the osteocyte is the mechanosensor, and that Wnt/Lrp5 is an important mechanism through which the osteogenic response is transduced. This proposition is supported by in vivo studies on Wnt reporter mice, where significant upregulation of the Wnt reporter Topgal was found in osteocytes 1 hr after an in vivo loading session, yet reporter activity was not detected in the surface cells (e.g., osteoblasts, lining cells) until 24 hrs after the stimulus.⁽⁴⁹⁾ Further, we⁽⁵⁰⁾ and others⁽⁵¹⁾ have reported that deletion of one or both β-catenin alleles from osteocytes impairs load-induced bone formation and/or mechanical adaptation.

Given that the regulation of Sost/sclerostin is clearly crucial for mechanotransduction in bone,⁽⁴⁸⁾ there has been great interest in understanding how Sost is mechanistically regulated by mechanical loading. Insight into this process was recently gleaned from clues provided by Van Buchem’s patients, who do not express SOST despite possessing an unperturbed SOST coding sequence and promoter. These patients are missing a small stretch of intergenic region, between Sost and its downstream neighbor Meox1. This long-range enhancer is crucial for Sost expression, and it contains consensus sequence for binding of the transcriptional repressor Mef2C. Conditional Mef2C deletion in bone recapitulates many of the HBM features of Sost deletion or Van Buchem’s effects.⁽⁵²⁾ A major effector of Mef2C activity is the class IIa histone deacetylases (Hdacs) 4 and 5.^(53,54) Hdac4 and Hdac5 prevent Mef2C-mediated Sost expression in osteocytes.⁽⁵⁵⁾ Recent experiments looking at the role of Hdacs in osteocyte mechanotransduction revealed that simultaneous genetic deletion of both Hdac4 and Hdac5 in osteocytes prevents Sost/sclerostin downregulation and load-induced bone formation.⁽⁵⁶⁾ Follow-up in vitro fluid flow experiments indicated that shear stress induces Hdac4/5 tyrosine phosphorylation at the focal adhesions, which triggers translocation of phosphorylated Hdac4/5 from focal adhesions to the nucleus, where these factors can interfere with Mef2C-driven Sost expression. At the focal adhesion complexes, focal adhesion kinase (Fak) appears to be required for Hdac processing and launch to the nucleus. Conversely, other studies have shown that mice with conditional deletion of Fak in osteoblasts/osteocytes have normal responsiveness to in vivo mechanical loading,⁽⁵⁷⁾ though successful deletion of Fak was never verified in these mice by protein or transcript reduction.

Another mechanically responsive Wnt signaling inhibitor is the Lrp5/6 antagonist Dkk1. Dkk1 antagonizes canonical Wnt signaling by binding to Lrp5/6 and Kremen (Krm), an accessory cell surface protein.⁽⁵⁸⁾ Formation of this complex (Lrp–Dkk1–Krm) induces the internalization and degradation of Lrp5/6 receptors. However, recent data indicates that when Lrps 5 and 6 are expressed at normal levels (and not overexpressed), Dkk1 can bind Lrps and inhibit Wnt/Lrp signaling even when Krm is absent or when the Krm binding sites on Dkk1 are mutated.⁽⁵⁹⁾ Thus the mechanisms of action for Dkk1’s inhibitory effect on Wnt signaling are unclear. Regardless of its mechanism of inhibition, Dkk1 has clear effects on bone mass. Mechanical stimulation downregulates Dkk1 expression in mouse bone^(47,60,61) and reduces circulating Dkk1 protein levels after exercise in young boys,⁽⁶²⁾ which suggests that (1) Dkk1 is mechanoresponsive across vertebrates, and (2) Dkk1 inhibition, like Sost, might be a mechanism that promotes canonical Wnt signaling during and after a mechanical stimulus.

Other, more poorly studied secreted inhibitors of Wnt signaling are also modulated by mechanical stimulation. For example, sFrp4 is a another extracellular Wnt antagonist, and both its expression level⁽⁶³⁾ and post-translational modification⁽⁶⁴⁾ in osteocytes are modulated by in vivo loading in rodents. However, no functional studies have been conducted to date, to address the necessity/dispensability of sFrp4. R-spondins (Rspos) are secreted factors that have been known for some time to enhance Wnt signaling activity, but their mode of action remained unclear as they were not capable of directly activating Wnt receptors. It turns out that all 4 members of the Rspo family (Rspo1-4) bind to (1) the single-pass transmembrane E3 ligases Rnf43 and Znrf3, and to (2) certain members of the leucine-rich repeat-containing G-protein coupled receptors (Lgrs), specifically, Lgr4, Lgr5, and Lgr6. Cell surface-bound Rnf43/Znrf3 normally interacts with activated Fzd-Lrp5/6 complexes, inducing polyubiquitination of the intracellular Fzd domains and subsequent internalization of the receptor complex, thereby inhibiting Wnt signaling. However, when Rspos and Lgr4/5/6 are present, Rspo sequesters Rnf43/Znrf3 away from the Fzd-Lrp5/6 complex and associates it with Lgr4/5/6, which induces internalization of the complex (Fig. 4). The Rspo-Lgr4/5/6 complex can be thought of as an “inhibitor of the inhibitor,” where Rnf43/Znrf3 is a potent inhibitor or Wnt signaling. Rspos appear to be mechanosensitive, and thus might provide another mechanism for enhancing Wnt signaling during mechanical stimulation. For example, Rspo1 is downregulated in the marrow of tail suspended mice (but not ovariectomized mice) and upregulated in mechanically stimulated cultured bone mesenchymal stem cells (BMSCs).⁽⁶⁵⁾ Interestingly, administration of Rspo1-producing adenovirus to tail suspended mice prevented bone loss, suggesting that the decrease in Rspo1 with disuse is physiologically important. In another study, cultured human BMSCs were subjected to a short period of mechanical vibration and the conditioned media was assayed for changes in secreted proteins using mass spectrometry.⁽⁶⁶⁾ Rspo1 was among the most highly upregulated factors in the media, which prompted the investigators to measure circulating Rspo1 levels in a young male volunteer after standing on a vibrating platform for 20 min. Serum Rspo1 was increased, as indicated by western blot (though n=1 and no controls were reported). Further, they administered recombinant Rspo1 to three different mouse models of aging and found increased bone formation. Thus, the Rspo and/or Rnf43/Znrf3 proteins might be additional targets for enhancing load-induced bone formation for therapeutic purposes. Conversely, the case for targeting Lgr4/5/6 receptors is less clear as other studies have suggested that Rspos can potentiate Wnt signaling independently of Lgrs. For example, Rspo2 inhibition affects limb development with or without the presence of Lgrs.⁽⁶⁷⁾ Other reports suggest that Rspos intervene in Wnt signaling through heparin sulfate proteoglycans (Hspgs)⁽⁶⁸⁾ rather than (or in addition to) Lgrs. This area remains controversial.

Figure 4:

[Left] Wnt ligands generate a trimeric complex consisting of Wnt, Lrp5, and Frizzledd (Fzd). [Middle] Activation of the complex induces the transmembrane ubiquitin ligases Znrf3 and Rnf43 to bind to the receptor complex and induce endocytosis, thereby limiting the strength of the Wnt signal. [Right] The presence or R-spondins (e.g., Rspo1) and its co-receptor Lgr4/5/6 induces a complex with Znrf3/Rnf43, sequestering Znrf3/Rnf43 away from the Lrp5/Fzd complex and consequently promoting the strength of the Wnt signal.

The discussion thus far has focused mainly on cell surface receptors/inhibitors and secreted inhibitors of Wnt signaling, but the real business end of Wnt signaling in mechanotransduction is the collection of Wnt proteins themselves. As there are 19 Wnts in the human and mouse genomes, it is an extremely difficult undertaking to sort out which Wnt or subset of Wnts are driving the activation of the pathway in mechanotransduction. One of the few studies to take a semi-comprehensive look at in vivo Wnt expression changes in mechanically stimulated bone tissue focused on a subset (15) of Wnts.⁽⁶⁹⁾ Wnt7b and Wnt1 increased 10- and 3-fold, respectively, several hours after mechanical stimulation of adult mouse tibiae. Whether those specific ligands are required for mechanical signaling, or whether other more modestly upregulated (or unchanged) Wnts can compensate if Wnt7b or Wnt1 are disabled, is still unknown. It has been much more lucrative to focus on co-receptors (e.g., there are only 2 Lrps that signal) though the Fzds are more difficult, with 10 family members, in both mouse and human. Identification of the exact Wnt milieu that is conducive to mechanical signaling in osteocytes (including functional studies that evaluate their requirement) will be the next mountain to climb in the field.

Summary

Mechanical loading is a simple yet effective way to increase bone mass, decrease bone loss, and improve bone strength. As the cellular and molecular mechanisms involved in bone cell mechanotransduction become better defined, new targets for therapeutic intervention will be identified. Modulation of the Wnt signaling pathway holds great promise in terms of altering bone mass and mimicking some of the positive effects of mechanical strain on bone. Eventually, as these and other pathways become more thoroughly characterized, it might be possible to trigger the signaling cascades activated by mechanical loading without applying any force to the bone. This would be particularly useful for patients in whom exercise would be beneficial to bone health, but have skeletal properties that would put them at too great a risk for fracture if exercise protocols were introduced.

Highlights.

• Wnt signaling is a key cellular mechanism in bone cells that facilitates structural adaptation in response to the mechanical environment.

• Secreted inhibitors of Wnt, transmembrane receptors and facilitators/modifiers, and Wnt-stimulated intracellular cascades are all critical for the proper response to mechanical loading.

• Newly identified components of the Wnt pathway have unexplored roles in bone cell mechanotransduction.