The osteocyte: doing the hard work backstage





Lynda F. BONEWALD, PhD
Matt PRIDEAUX, PhD
Oral Biology, School of Dentistry University of Missouri-Kansas City, USA

The osteocyte: doing the hard work backstage


by M. Pr ideaux and L. F. Bonewald, USA



Osteocytes are the most numerous and long-lived of all bone cells; however, relatively little is known about their function when compared with osteoblasts and osteoclasts. Although originating from osteoblast precursors, they display dramatic differences in morphology and gene expression, hence suggesting their functions differ from those of osteoblasts. So far, roles have been determined for the osteocyte in processes such as mechanotransduction and bone homeostasis, via modulation of osteoblast and osteoclast activity. In addition, the osteocyte network has been shown to act as part of an endocrine system, targeting organs such as the kidney and skeletal muscle. Expression of phosphate regulatory genes by osteocytes controls phosphate metabolism within and beyond bone and plays a key role in mineralization of the bone extracellular matrix. Osteocytes are also capable of expressing markers of bone resorption and can form new bone matrix, suggesting that they are capable of remodeling their microenvironment. Clearly, the osteocyte, far from being a passive cell trapped within the mineralized bone matrix plays a highly active and functional role in the maintenance of bone strength and viability.

Medicographia. 2012; 34:228-235 (see French abstract on page 235 )



Although the biology and function of both osteoblasts and osteoclasts have been well documented, the osteocyte remains more of a mystery. For years, the study of osteocytes has been stymied by their location within the mineralized bone matrix and their relative inaccessibility, compared with the cells situated on the bone surface. In addition, their relative lack of abundance of cellular organelles such as the Golgi apparatus and endoplasmic reticulum,1 when compared with osteoblasts and osteoclasts,2 previously led to the assumption that these cells were metabolically inactive and of little importance during bone growth and development. The multiple roles of the osteocyte, both within and beyond the bone microenvironment are, however, starting to be revealed. The ability to delete genes specifically within osteocytes in animal models, coupled with the development of several osteocyte-like cell lines,3-5 has generated increased interest in these once forgotten cells. No longer merely considered as “placeholders” within the bone matrix, osteocytes have been shown to exhibit complex functions that are both numerous and vital in the development and maintenance of bone health. This review will focus on these functions in light of recent discoveries, which demonstrate the importance of the osteocyte as an orchestrator of bone modeling and remodeling and as part of an endocrine system, targeting organs outside of the bone environment.


Figure 1
Figure 1. The process of osteoblast-toosteocyte
differentiation.

(A) Tetrachrome staining of murine cortical bone. The osteoid seam is demonstrated with light blue staining and the mineralized bone is stained black with von Kossa. The stages of differentiation are described as follows: 1) A mature osteoblast on the surface of the osteoid. 2) An osteoid-osteocyte, which is embedded in the unmineralized osteoid. 3) A mineralizing osteocyte, which is partially surrounded by mineral. 4) A mineralizing osteocyte, completely surrounded by mineral. 5) A mature osteocyte, embedded deep within the mineralized extracellular matrix. (B) A schematic diagram showing the differentiation process outlined in (A) and the expression of known genes at each of these stages of differentiation. The numbers in brackets correspond to the numbers in (A).


Osteocyte differentiation and morphology

While it has been known for decades that osteocytes are descended from terminally differentiated osteoblasts,1 the mechanisms that govern this process of differentiation are still poorly understood. The morphological changes associated with this transition have, however, been well characterized6 and are summarized in Figure 1. During differentiation, the osteoblast changes from a polygonal morphology toward a dendritic appearance, accompanied by the development and elongation of numerous cellular projections, or processes. Concurrently, there is a reduction in cell volume (of up to 70%) and cellular organelles as the cell becomes embedded within the bone matrix. This embedding cell, termed an osteoid-osteocyte,6 is responsible for mineralizing its surrounding extracellular matrix (ECM)7 and exhibits polarity with regard to its process formation6 as it further differentiates into a mature osteocyte. Osteocyte differentiation has commonly been regarded as a passive process, whereby an osteoblast slows its matri-synthesizing capacity and becomes buried by the matrix produced by its neighboring cells.8 Research by others, however, has suggested that the embedding of an osteoid-osteocyte is an active process, as demonstrated by the requirement of collagenase activity for the formation of cell processes and the development of the lacunocanalicular system.9,10 In particular, modification of the ECM by membrane type 1 matrix metalloproteinase (MT1-MMP) appears to be essential for development of the cell processes.9 In addition, recent studies have demonstrated that osteocytes embedded within the bone have the capacity to extend their processes and indeed, are capable of forming new connections with neighboring osteocytes and osteoblasts.11 This therefore suggests, that rather than simply being a static cell within the mineralized bone matrix, osteocytes are, in fact, highly dynamic.





Until recently, characterizing the molecular and genetic changes that an osteoblast undergoes as it differentiates into an osteocyte has proved challenging due to the lack of specific osteocyte marker genes. Such genes are, however, now being identified. The onset of expression of genes such as E11/ gp38 (E11), fibroblast growth factor 23 (FGF23), and sclerostin (SOST), concurrent with the up regulation of dentin matrix protein 1 (DMP1), phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX), and matrix extracellular phosphoglycoprotein (MEPE), and the down regulation of alkaline phosphatase and type I collagen are all indicative of transition toward an osteocyte phenotype.12 Such genes have functions ranging from the regulation of mineralization (DMP1), phosphate homeostasis (PHEX, MEPE, FGF23), and cytoskeletal arrangement and process development (E11/ gp38), which will be discussed later in this review.

Although the mature osteocyte is surrounded by a mineralized matrix and may therefore appear isolated from its neighboring cells both within the matrix and on the bone surface, these cells, in fact, display a high degree of connectivity, as is demonstrated in Figure 2.

The cell processes, which lie within the narrow canaliculi, connect osteocytes to other osteocytes, osteoblasts, and lining cells via gap junctions.6,13 This unique morphology of the osteocyte allows for the passage of nutrients and biochemical signals from one cell to the next and, as such, forms a functional network of cells, facilitating communication and maintaining cell viability.14 Indeed, proper osteocyte function is dependent on a viable network of cells and disruption of this network can have devastating consequences for bone health.


Figure 2
Figure 2. Scanning electron microscopy of bone microstructure.

(A) Scanning electron microscopy of mouse cortical bone, showing osteocyte lacunae (arrowheads) and blood vessels (BV). The osteocytes appear isolated from each other and the blood vessels. (B) Scanning electron microscopy of the same area of cortical bone after acid-etching of the bone surface. The same osteocyte lacunae are marked by arrowheads as in (A). (C) A higher-magnification image of the area outlined by a white box in (B). The degree of connectivity between the processes of the osteocytes with other osteocytes and the blood vessels is readily apparent. An osteocyte in close proximity to a blood vessel is marked by an asterisk. (D) A higher magnification image of the red box in (B) showing an occupied lacuna (arrowhead), connected to other osteocytes and the bone surface via numerous processes.



Figure 3
Figure 3. Regulation of bone resorption and formation via osteocytes.

Osteocyte (OCY) expression of RANKL and M-CSF promotes, whereas the expression of OPG inhibits, osteoclast (OC) activity and subsequent bone resorption. Osteocytes also secrete factors that activate Wnt/-catenin signaling in osteoblasts (OB), such as PGE2, NO, and ATP, to promote bone formation. However, osteocytes also release factors such as sclerostin, DKKs, and SFRPs, which have an inhibitory effect on Wnt/β-catenin signaling and result in decreased osteoblast activity.


Osteocyte functions – regulators of bone modeling

As previously discussed, osteocytes are, via their processes, connected to osteoblasts on the bone surface. This connectivity suggests a role for the osteocyte in regulating osteoblast activity.2 It has previously been demonstrated that conditioned media from MLO-Y4 osteocyte-like cells15 and primary chick osteocytes16 enhances early osteoblast differentiation and alkaline phosphatase activity. Osteocytes in vivo are also known to recruit mesenchymal stem cells to fracture sites, via secretion of osteopontin.17 These data would suggest positive regulation of osteoblast activity via the osteocyte; however, it is the ability of these cells to negatively control bone formation that is receiving the most attention. The Wnt signaling pathway plays an important role in promoting early osteoblast differentiation and osteocytes express known inhibitors of the Wnt signaling pathway such as the dickkopf-related proteins (DKKs), secreted frizzled-related proteins (SFRPs), and sclerostin.18 Inhibition of Wnt signaling either by direct binding to Wnt ligand (SFRP1), or binding of the coreceptor LRP5/6 (DKK1, sclerostin) results in phosphorylation of β-catenin by glycogen synthase kinase 3-β(GSK3-β) and subsequent degradation by the proteasome.18 Therefore, β-catenin is unable to translocate to the nucleus and activate the transcription factors required for inducing osteoblast differentiation and subsequent bone formation. While all of these factors are known to target the Wnt pathway, it is the activity of sclerostin that is garnering the most interest. Since its discovery as the secreted protein product of the SOST gene, which was identified by its absence in patients suffering from the sclerosing bone disorders van Buchem disease and sclerosteosis,19,20 sclerostin has emerged as one of the most important therapeutic targets for bone disorders. Inactivating mutations in the SOST gene leads to an increase in bone mass and resistance to fracture.21 Using this knowledge, specific targeting of sclerostin catabolic activity using monoclonal antibodies has demonstrated beneficial effects in animal models and human trials.22 Recent studies have also suggested that inhibiting sclerostin activity can aid with fracture healing23 and that the anabolic effects observed with parathyroid hormone (PTH) treatment are mediated by down-regulation of SOST expression.24,25

In addition to regulation of osteoblast activity, there is increasing evidence to suggest a role for the osteocyte during osteoclastogenesis. The differentiation of a mature osteoclast requires binding of receptor activator of nuclear factor κB ligand (RANKL), found on the surfaces of cells of the osteoblast lineage, to its receptor, RANK, expressed by osteoclast precursors.26 RANKL expression has been demonstrated in osteocytes, along with expression of the decoy receptor, osteoprotegerin (OPG).27,28 The ratio of RANKL/OPG expression is responsible for regulation of osteoclast development and activity. Previous studies have demonstrated that MLO-Y4 osteocyte- like cells support the activation of osteoclasts in vitro and express RANKL and OPG.28 However, others have suggested that osteocytes only induce osteoclast formation and activity when undergoing apoptosis.29 Additionally, mice in which the diphtheria toxin receptor is conditionally expressed in osteocytes, display dramatic increases in osteoclast number and activity following osteocyte death after diphtheria toxin injection,30 suggesting that osteocyte death induces osteoclastogenesis. Conversely, stimulation of osteocytes by mechanical loading has been shown to increase OPG expression and decrease osteoclastogenesis,31 suggesting a dual role for osteocytes in the regulation of bone resorption. Interestingly, deletion of β-catenin specifically in osteocytes in mice using the DMP1-Cre system, resulted in significantly decreased OPG expression and enhanced osteoclast activity.32 These mice were characterized by a dramatic reduction in both cancellous and cortical bone volume, with no effect on osteoblast or osteocyte number or viability. These results suggest an important role for the Wnt signaling pathway in osteocytes in the negative regulation of bone resorption. The importance of osteocyte signaling in both bone formation and resorption is summarized in Figure 3.

Mechanotransduction

The idea of a “mechanostat”, a sensor of mechanical loading within the bone, was first proposed by Harold Frost in 1987.33 This mechanostat would have the ability to sense deformation of the bone due to mechanical stress and regulate changes in bone mass, accordingly. The osteocyte, within the mineralized bone would appear to be ideally located to sense such changes in bone loading and, because of its unique cellular morphology, be able to communicate such strains to osteoblasts and osteoclasts. Indeed, osteocytes have been shown to respond to fluid flow shear stress (FFSS)34 and membrane stretching,35 two different mechanisms inducing deformation of the osteocyte dendrites or/and cell body in vitro. Ex vivo, it has been shown that deformation of the osteocyte lacunae and canaliculi occurs in response to mechanical loading of cortical bone,36 and osteocyte predominant genes such as the DMP1, E11/gp38, MEPE, and sclerostin genes have been shown to be regulated by mechanical loading in vivo. Until recently, however, it was unknown how the osteocyte was able to sense this mechanical stress but recent studies have suggested that primary cilia, which play a mechanosensory role in many cell types, are known to be expressed by osteocytes.37 Moreover, deletion of Pkd1, an integral component of the cilia signaling pathway, attenuated increases in bone mass observed in mechanically loaded mice,38 suggesting the importance of the cilia in modulating the response of osteocytes to load.

The importance of the osteocyte processes in sensing strain induced by fluid flow was demonstrated in a recent study by Burra et al.39 It was observed that disruption of the glycocalyx— the protective coating of glycoproteins secreted by the cell—of the processes diminished the ability of the osteocytes to sense and respond to FFSS. No such effect was observed when the glycocalyx of the cell body was similarly disturbed, suggesting that the processes, and not the cell body, are responsible for detecting mechanical strain.

Whichever way mechanical loading is sensed by osteocytes, it needs to be translated into biochemical signals to induce an appropriate biological response. The Wnt/β-catenin signaling pathway has been widely implicated in modulating the anabolic effects of mechanical loading and the catabolic effects of unloading (for review, see reference 18). In vivo loading of bone results in decreased production of sclerostin by osteocytes, promoting Wnt/β-catenin signaling, whereas unloading increases sclerostin expression. Regulation of bone mass by estrogen signaling has also been suggested, as translocation of estrogen receptor α(ERα) to the nucleus was observed in osteocytes in response to mechanical strain.40 Such translocation was found to be necessary for transportation of β-catenin to the nucleus in osteoblasts,41 suggesting crosstalk between ERα and the Wnt signaling pathway. This therefore indicates a mechanism for postmenopausal bone loss, whereby a decline in circulating estrogen levels leads to decreased expression of ERα and, subsequently, an attenuated response to mechanical loading via Wnt/β-catenin signaling.

It is also known that mechanical strain leads to the rapid release of factors such as prostaglandin E2 (PGE2)35,36 and nitric oxide (NO).34,35,42 Release of PGE2 in response to mechanical strain has been demonstrated to be dependent on connexin 43 hemichannels,43 with the opening of these hemichannels essential for the passage of PGE2 and other soluble factors between cells. PGE2 has recently been shown to promote β-catenin nuclear translocation in osteocytes and this occurs via inactivation of GSK3-β,43 suggesting a regulatory role for prostaglandin signaling on the Wnt/&bta;-catenin pathway. NO is also believed to play a similar regulatory role, as inhibitors of NO synthase attenuate the stabilization of β-catenin observed after mechanical stimulation and prevent the activation of Wnt target genes.44

Phosphate homeostasis and matrix mineralization

The importance of the osteocyte in the regulation of phosphate homeostasis has been clearly demonstrated in diseases such as X-linked hypophosphatemic rickets (XLH) and autosomal dominant hypophosphatemic rickets (ADHR), in which defects in osteocyte-specific or predominant proteins result in decreased circulating levels of inorganic phosphate (Pi). FGF23, a phosphaturic hormone that is synthesized primarily by osteocytes, inhibits reabsorption (and therefore increases excretion) of phosphate by the kidney and prevents phosphate uptake in the intestine (for review, see reference 45). FGF23 expression can be regulated by diet, 1,25-dihydroxyvitamin D3 levels, and circulating PTH. In addition, other osteocytesecreted proteins are known to regulate FGF23 activity and, as a consequence, serum Pi levels. Inactivating mutations in PHEX such as those observed in the hypophosphatemic (HYP) mouse model, result in increased circulating FGF23 levels and hypophosphatemia,45,46 and deletion of FGF23 is able to reverse the HYP phenotype.46 PHEX has been shown to co-localize with FGF23 in osteocytes,45 although the mechanism by which PHEX regulates FGF23 remains to be fully elucidated.

MEPE is another osteocyte-secreted protein that is responsible for elevating FGF23 levels. MEPE is not known, however, to act on FGF23 directly but instead induces hypophosphatemia via inhibition of PHEX enzymatic activity.45 Cleavage of MEPE by cathepsin B releases the ASARM peptide, which, in addition to antagonizing PHEX, can bind directly to hydroxyapatite to inhibitmineralization.47 DMP1 is another small integrinbinding ligand N-linked glycoprotein (SIBLING) that is produced by osteocytes and regulates Pi upstream of FGF23.46,48 Dmp1-null mice show increased FGF23 levels in osteocytes, decreased serum Pi levels, and have an osteomalacic phenotype.48 In addition, these mice display defective ECM mineralization around the osteocyte lacunae and impaired osteoblast- to-osteocyte differentiation.48

These data suggest the importance of DMP1, not only in Pi homeostasis but also in ECM mineralization and indicate a role for the early osteocyte in mineralizing its surrounding matrix. In addition, preosteocytes in vitro and in vivo have been shown to initiate mineralization by depositing calcospherulites along collagen fibrils,7 and mineralization of primary osteoblasts cultured in vitro was found to be associated with cells that were expressing osteocyte markers.11 All in all, these results suggest both localized and endocrine roles for the osteocyte in regulating phosphate homeostasis.

Microenvironment remodeling by osteocytes

Much debate has occurred regarding the ability of an osteocyte to resorb bone from its perilacunar surface and therefore remodel its surrounding microenvironment. Initially suggested as a mechanism for transiently increasing the bioavailability of calcium,49 “osteocytic osteolysis” has been reported in response to space flight in rats,50 and PTH administration in rats induced an increase in osteocyte organelle number and activity, concomitant with osteolysis.51 Others, however, have denied such activity, claiming that osteocytes do not have the capacity to remodel their lacunae.52

Improved histomorphometric analysis, combined with the advent of molecular biology techniques has enabled further investigation into this area and evidence is growing to suggest the ability of the osteocytes to remodel their surrounding matrix. Continuous administration of PTH was found to induce osteocyte expression of acid phosphatase and increase osteocyte lacunar area.53 In addition, increases in lacunar size and expression of osteoclastmarker genes such as acid phosphatase and cathepsin K were observed in lactating rats.54 These results suggest a mechanism whereby supplemental sources of calcium can be utilized by osteocytes during periods when excess calcium is required.

In addition to the removal of their lacunar matrix, osteocytes have also been demonstrated to synthesize new bone matrix. Tetracycline labeling has been observed around osteocyte lacunae and canaliculi in response to decreased PTH55 and deposition of new matrix was observed in the osteocyte lacunae of egg-laying hens.56 The participation of osteocytes in such remodeling is a further indication of the activity of these cells. Unlike the substantial bone resorbing capabilities of the osteoclast, the role of the osteocyte in such remodeling is likely to enable the transient mobilization of factors and minerals stored within the ECM. The lacunocanalicular system would allow the rapid transport of such factors to their required targets. It has long been known that many bioactive proteins and minerals are stored within the ECM. The osteocytes may therefore act as the “gatekeepers” for these factors, allowing access to them when required.

Perspective

For a cell that was once considered to act as little more than a “placeholder” within the bone matrix, the diverse functions of the osteocyte demonstrate a dynamic, active role for this cell, both within and outside the bone environment. Recent research has identified novel functions of the osteocyte, such as control of phosphate homeostasis and osteoclastogenesis, while confirming long-held hypotheses such as osteocytic osteolysis and the ability to sense mechanical strain.

However, these studies may only be the “tip of the iceberg” as regard osteocyte activity. Recently, it has been shown that osteocytes may regulate the differentiation of skeletal muscle cells by the release of soluble factors57 and that factors released by muscle cells may influence osteocyte activity and viability,58 suggesting cross-talk between bone and muscle. In addition, the expression of osteocyte-specific marker genes have been observed in calcified regions of the aorta, suggesting that differentiation of vascular smooth muscle cells toward an osteocyte phenotype may promote pathological calcification. 59

Although, clearly, there is still much to learn about the osteocyte, it is becoming apparent that it shares equal importance with its more illustrious neighboring bone cells. Future therapeutics, rather than directly targeting osteoblast or osteoclast activity, could instead be directed against osteocytes to regulate bone mass. Indeed, targeting of sclerostin activity using such treatments has already proved successful. It is, therefore, time for the osteocyte to move from the back of the stage to the center and share the limelight that its crucial role deserves. _


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Keywords: bone; mechanotransduction; mineralization; osteocyte; phosphate