osteocytes , which are embedded in the bone matrix , establish an extensive intracellular and extracellular communication system via gap - junction - coupled cell processes and canaliculi through which cell processes pass throughout the bone and the communication system is extended to osteoblasts on the bone surface ( marks and odgren 2002 ) .
bone tissue is able to adapt its mass and three - dimensional structure to the prevailing mechanical usage to achieve higher load - bearing efficiency ( wolff 1892 ) .
the lacunocanalicular network formed by osteocytes is thought to be an ideal mechanosensory system and suitable for mechanotransduction by which mechanical energy is converted into electrical and/or biochemical signals ( burger and klein - nulend 1999 ; martin 2000 ; ehrlich and lanyon 2002 ; knothe tate 2003 ; bonewald and johnson 2008 ; noble 2008 ) .
however , the special anatomic features of osteocytes , which are isolated in lacunae but are three - dimensionally connected to each other , make it difficult to study their function .
this review focuses on the mechanism of osteocyte apoptosis - induced bone resorption and functions of the osteocyte network in the regulation of bone mass under physiological and unloaded conditions .
osteocyte death occurs during aging , after menopause , at unloading and under pathological conditions such as microcracks and the death of osteocytes is closely coupled with bone resorption ( noble and reeve 2000 ; cardoso et al .
cells die mainly through one of three pathways , i.e. , apoptosis , autophagic cell death and necrosis , under physiological and pathological conditions , although dying cells can share apoptotic and autophagic features ( bursch 2001 ; fig .  1 ) .
if cells die through apoptosis or autophagy , death is completed with the removal of the cells through engulfment by scavengers . in these cases ,
the cells are quietly removed without inflammation , because the integrity of the cytoplasmic membranes is maintained when phagocytosis occurs .
in contrast , strong insults cause bioenergetic failure and rapid loss of the integrity of the cytoplasmic membrane , both of which are core events of necrosis .
necrosis leads to the rupture of the cytoplasmic membrane and most of the intracellular content is released into the extracellular environment .
the released intracellular content encompasses immunostimulatory molecules including the so - called damage - associated molecular pattern molecules , such as s100 family molecules , high - mobility group box 1 protein , purine metabolites , heat - shock proteins and uric acid ( lotze and tracey 2005 ; zong and thompson 2006 ) .
the release of the immunostimulatory molecules actively recruits a defensive or a reparative response to the damaged regions .
if cells at the terminal phase of apoptosis or autophagic cell death are not engulfed by phagocytes , a transition to necrosis ensues and the cells are eliminated by cell disruption .
this process is called secondary necrosis ( majno and joris 1995 ; silva et al .
2008 ) . as scavengers can not reach osteocytes , any type of osteocyte death will end in the rupture of the cytoplasmic membrane .
after cell rupture , immunostimulatory molecules are released from lacunae through canaliculi to the bone surface and vascular channels and facilitate the recruitment and activation of macrophages , thereby promoting the production of proinflammatory cytokines , including tumor necrosis factor- ( tnf- ) , interleukin ( il)-6 and il-1 ( lotze and tracey 2005 ) .
further , il-6 and il-1 induce receptor activator of nuclear factor -b ligand ( rankl ) expression , which is essential for osteoclastogenesis ( yasuda et al .
il-6 and il-1 are the most important proinflammatory cytokines triggering inflammatory bone loss ( firestein 2003 ) , primary and secondary osteocyte necrosis could lead to the enhancement of osteoclastogenesis and bone resorption , which comprise a repair process in bone ( fig . 
cells die mainly through one of three pathways : apoptosis , autophagic cell death and necrosis . as osteocytes at the terminal phase of apoptosis or autophagic cell death are not engulfed by phagocytes , the process of secondary necrosis ensues . after cell rupture ,
immunostimulatory molecules including high - mobility group box 1 ( hmgb1 ) , s100 , heat shock protein ( hsp ) , adenosine triphosphate ( atp ) and uric acid are released from lacunae through canaliculi to the bone surface and vascular channels , where they facilitate the recruitment and activation of macrophages , thereby promoting the production of proinflammatory cytokines , including tumor necrosis factor- ( tnf- ) , interleukin 6 ( il-6 ) and interleukin 1 ( il-1 ) , which induce osteoclastogenesis and bone resorption .
apoptotic osteocytes release osteoclastogenesis - promoting factors , including soluble receptor activator of nuclear factor -b ligand ( srankl ) , il-6 and soluble il-6 receptor ( sil-6r ) and induce tnf- expression in osteoclast precursors in vitro osteocyte death and bone resorption .
cells die mainly through one of three pathways : apoptosis , autophagic cell death and necrosis . as osteocytes at the terminal phase of apoptosis or autophagic cell death are not engulfed by phagocytes , the process of secondary necrosis ensues . after cell rupture ,
immunostimulatory molecules including high - mobility group box 1 ( hmgb1 ) , s100 , heat shock protein ( hsp ) , adenosine triphosphate ( atp ) and uric acid are released from lacunae through canaliculi to the bone surface and vascular channels , where they facilitate the recruitment and activation of macrophages , thereby promoting the production of proinflammatory cytokines , including tumor necrosis factor- ( tnf- ) , interleukin 6 ( il-6 ) and interleukin 1 ( il-1 ) , which induce osteoclastogenesis and bone resorption .
apoptotic osteocytes release osteoclastogenesis - promoting factors , including soluble receptor activator of nuclear factor -b ligand ( srankl ) , il-6 and soluble il-6 receptor ( sil-6r ) and induce tnf- expression in osteoclast precursors in vitro apoptotic osteocytes show enhancement of osteoclastogenesis in vitro ( kogianni et al .
addition of apoptotic bodies from osteocytes but not osteoblasts induces osteoclastogenesis through the induction of tnf- production by mononuclear osteoclast precursors ( kogianni et al .
conditioned medium from apoptotic osteocytic cell line mlo - y4 contains more soluble rankl than that from non - apoptotic mlo - y4 cells and induces osteoclastogenesis ( al - dujaili et al . 2011 ) .
methotrexate ( mtx)-induced apoptosis of mlo - y4 cells increases il-6 and il-11 expression and the conditioned media promote osteoclastogenesis ( shandala et al . 2012 ) .
further , apoptotic mlo - y4-conditioned media promote osteoclast precursor adhesion to endothelial cells via intercellular adhesion molecule 1 ( icam-1 ) , probably through the induction of il-6 and soluble il-6 receptor ( cheung et al .
2012 ) . therefore , apoptotic osteocytes can upregulate the expression of osteoclastogenesis - promoting factors ( fig .  1 ) . another special feature of osteocyte death is the sustained detection of degraded dna by terminal deoxynucleotidyl transferase - mediated dutp nick end - labeling ( tunel ; weinstein et al .
this indicates that a part of the dna degraded during the process of apoptosis is retained in the lacunae , even after secondary necrosis . even during primary necrosis , randomly degraded dna retained in the lacunae can be detected by tunel .
the reactivity of tunel in the lacunae is retained until the bone , including the lacunae , is resorbed .
as the death of osteocytes induces bone resorption , the physiological function of osteocytes has been considered to be the inhibition of bone resorption ( gu et al .
2007 ) . transgenic mice expressing diphtheria toxin receptor under the control of the dentin matrix protein 1 ( dmp1 ) promoter show enhanced bone resorption resulting in severe osteoporosis after the injection of diphtheria toxin .
dmp1 expression begins in osteoblasts when they are about to be embedded into the bone matrix and dmp1 expression is high in immature osteocytes but decreases during osteocyte maturation ( toyosawa et al .
the intracellular content of osteocytes is released from the lacunae onto the bone surface and vascular channels and promotes osteoclastogenesis as a process of repair ( fig .  1 ) .
this seems to be the case , because rankl is highly induced after the injection of diphtheria toxin ( tatsumi et al .
although osteocyte death causes bone resorption , whether live osteocytes inhibit bone resorption remains unclear .
gja1 ( connexin 43 ) is the major gap junction protein and plays important roles in osteogenesis and osteoblast function ( lecanda et al .
2011 ) . targeted deletion of gja1 in osteocytes results in the disruption of the intercellular communication of osteocytes through gap junctions , while retaining communication through the canalicular network ( bivi et al .
osteocyte apoptosis is increased , the osteoclast number and surface are increased at the endocortical surface and the marrow cavity is enlarged .
this indicates that osteocyte apoptosis in conditional gja1 knockout mice could induce bone resorption , probably because the intracellular content of dead osteocytes could be released through the intact canalicular network and trigger osteoclastogenesis and bone resorption ( fig .  1 ) .
the abundance of osteoclast precursor cells and immune cells in bone marrow should facilitate bone resorption at the endocortical surface after osteocyte apoptosis .
another model of dysfunction of the osteocyte network is the osteoblast - specific bcl2 transgenic mouse . bcl2 transgenic mice have a reduced number of osteocyte processes probably because of the function of bcl2 to alter cytoskeletal organization ; the osteocytes gradually die by apoptosis and the frequency of tunel - positive lacunae reaches 80% at 4  months of age ( moriishi et al .
however , bone resorption is not induced by osteocyte apoptosis in bcl2 transgenic mice and both the intracellular and extracellular osteocyte networks are disrupted at 4  months of age by the reduced number of canaliculi and the accumulation of tunel - positive lacunae .
the absence of enhanced bone resorption in bcl2 transgenic mice seems to be attributable to the failure of canalicular ( extracellular ) network formation , which interrupts the release of the intracellular content of dead osteocytes onto the bone surface and into vascular channels .
contrary to many previous observations that osteocyte apoptosis enhances bone resorption , the number of osteoclasts and bone resorption are reduced in bcl2 transgenic mice .
interestingly , the number of osteoclasts is reduced on the endocortical surface but not on the periosteal surface in cortical bone , suggesting that the osteocyte network plays a major role in regulating osteoclastogenesis on the endocortical surface of cortical bone .
these findings indicate that the disruption of both the intercellular and extracellular communication systems of osteocytes suppresses osteoclastogenesis and bone resorption . as bcl2 overexpression in osteoblasts
2011 , 2012 ) , we conclude that the osteocyte network enhances osteoclastogenesis and bone resorption under physiological conditions ( fig . 
the osteocyte network mildly stimulates osteoclastogenesis and bone resorption and mildly inhibits osteoblast function and bone formation .
rankl expression in osteoblasts and sclerostin ( sost ) expression in osteocytes are , at least in part , responsible for the enhanced bone resorption and suppressed bone formation , respectively . both rankl and osteoprotegerin ( opg )
are highly expressed in osteocytes and opg seems to be trapped by rankl on the surface of osteocytes .
srankl secreted by osteocytes might be involved in the regulation of osteoclastogenesis ( dashed arrow ) .
the osteocyte network strongly stimulates osteoclastogenesis and bone resorption and strongly inhibits osteoblast function and bone formation .
the osteocyte network enhances rankl expression in osteoblasts and sost expression in osteocytes in the unloaded condition .
upregulation of rankl in osteocytes in the unloaded condition is controversial . in a , b ,
the thickness of the lines and arrows reflects the strength of the effects and the height of the osteoblasts reflect their activities regulation of bone mass by the osteocyte network .
the osteocyte network mildly stimulates osteoclastogenesis and bone resorption and mildly inhibits osteoblast function and bone formation .
rankl expression in osteoblasts and sclerostin ( sost ) expression in osteocytes are , at least in part , responsible for the enhanced bone resorption and suppressed bone formation , respectively . both rankl and osteoprotegerin ( opg )
are highly expressed in osteocytes and opg seems to be trapped by rankl on the surface of osteocytes .
srankl secreted by osteocytes might be involved in the regulation of osteoclastogenesis ( dashed arrow ) .
the osteocyte network strongly stimulates osteoclastogenesis and bone resorption and strongly inhibits osteoblast function and bone formation .
the osteocyte network enhances rankl expression in osteoblasts and sost expression in osteocytes in the unloaded condition .
upregulation of rankl in osteocytes in the unloaded condition is controversial . in a , b ,
the thickness of the lines and arrows reflects the strength of the effects and the height of the osteoblasts reflect their activities osteocytes highly express rankl compared with osteoblasts ( nakashima et al .
conditional rankl deletion by using dmp1 cre transgenic mice results in increased bone mass ( nakashima et al .
rankl is produced as a membrane - bound or soluble form , although the membrane - bound form is much more efficient in terms of osteoclast formation than the soluble form ( lum et al .
the production of soluble rankl ( srankl ) in mlo - y4 cells is controversial ; one study has shown that the conditioned media from mlo - y4 cells support osteoclastogenesis , whereas another has demonstrated that conditioned media fail to support osteoclastogenesis ( zhao et al .
2011 ) . as srankl is detected in the circulatory system of humans ( schett et al . 2004
2005 ) , the physiological importance of srankl produced by osteocytes needs to be investigated , because the direct interaction of osteocytes with osteoclasts and their precursors will be limited ( fig .  2 ) .
the secreted factor osteoprotegerin ( opg ) , which is produced in osteocytes , might also be released onto the bone surface and vascular channel through canaliculi without being trapped by the rankl on the surface of osteocytes in the rankl conditional knockout mice , because opg is also highly expressed in osteocytes ( moriishi et al .
most of the opg produced by osteocytes seem to be trapped by rankl on the surface of osteocytes , because the osteocyte network enhances osteoclastogenesis under physiological conditions ( moriishi et al .
ablation of osteocytes by diphtheria toxin severely reduces bone formation , indicating that osteocyte death also affects bone formation .
reduced bone formation also seems to be caused by secondary necrosis - induced inflammation , because osteoblast maturation is severely inhibited in diphtheria toxin receptor transgenic mice ( tatsumi et al .
in contrast , osteocyte density is negatively correlated with bone formation and both the empty lacunar density and periosteal bone apposition increase with age , suggesting a linkage between the two phenomena ( lazenby 1990 ; qiu et al .
2002 ; metz et al . 2003 ; russo et al . 2006 ; hedgecock et al . 2007 ) .
mice carrying a targeted mutation of col1a1 , encoding a collagenase - resistant form of type i collagen , exhibit osteocyte apoptosis and increased bone formation ( zhao et al .
further , enhanced bone formation at the periosteal and endocortical surfaces is localized in the area in which viable osteocytes are lost in osteocyte - specific gja1 knockout mice ( bivi et al .
2012 ) . in a study of bcl2 transgenic mice at 4  months of age , bone formation in both trabecular and cortical bones was enhanced when the osteocyte network was disrupted , although the contribution of bcl2 overexpression in osteoblasts to bone formation could not be completely excluded ( moriishi et al .
2012 ) . combining the findings from these mouse models with the histological observations showing the inverse linkage between osteocyte density and bone formation ,
we conclude that the osteocyte network suppresses bone formation under physiological conditions ( fig .  2 ) .
sost ( sclerostin ) is specifically expressed in osteocytes and inhibits osteoblast function and bone formation by antagonizing canonical wnt signaling through binding to wnt co - receptor low density lipoprotein receptor - related protein ( lrp ) 5 and lrp6 , and bone formation is enhanced in trabecular bone and on the endocortical and periosteal surfaces of cortical bone in sost - deficient mice ( winkler et al .
2004 , 2007 ; bellido et al . 2005 ; li et al . 2005 , 2008 ; poole et al . 2005 ; semenov et al .
further , osteocyte - specific deletion of the parathyroid hormone ( pth ) receptor increases sost expression and the specific expression of constitutively active pth receptor in osteocytes reduces it , indicating the involvement of sost in the anabolic action of pth ( powell et al .
2011 ) . therefore , sost is a major negative regulator of osteoblast differentiation and function produced by osteocytes . as the dissemination of sost through canaliculi
is impaired in bcl2 transgenic mice , impaired secretion to the bone surface should greatly contribute to enhanced bone formation ( moriishi et al .
2012 ) . however , bone formation is enhanced in trabecular bone and on the periosteal surface but not on the endocortical surface of cortical bone in bcl2 transgenic mice ( moriishi et al .
thus , the osteocyte network might send some positive signals to osteoblasts on the endocortical surface , in addition to the negative signal by sost .
application of mechanical stimuli to osteocytes in vitro induces the release of anabolic factors , such as prostaglandin e2 , prostaglandin i2 , nitric oxide , cyclooxygenase-2 and endothelial nitric oxide synthase ( burger and klein - nulend 1999 ) .
further , it is widely accepted that mechanical stress increases bone mass by enhancing bone formation and inhibiting bone resorption , whereas unloading reduces bone mass by inhibiting bone formation and enhancing bone resorption and that the osteocyte network is an appropriate system for the sensing apparatus ( burger and klein - nulend 1999 ; martin 2000 ; ehrlich and lanyon 2002 ; knothe tate 2003 ; noble 2008 ; bonewald and johnson 2008 ) .
however , difficulties have been experienced in establishing that the osteocyte network is responsible for the regulation of bone mass by mechanical stress , because osteocyte death provokes bone resorption .
bone - specific gja1 conditional knockout mice , in which intercellular communication through gap junctions is disrupted , represent a potential mouse model for evaluating whether the osteocyte network acts as the mechanosensing apparatus .
however , the responses to mechanical stress in the gja1 conditional knockout mice are variable . in the unloaded condition following hind limb muscle paralysis ,
bone resorption is enhanced on the endocortical surface of tibiae in wild - type mice but not in the conditional gja1 knockout mice , in which gja1 is deleted in osteoblasts and osteocytes using 2.3-kb col1a1 promoter cre transgenic mice ( grimston et al . 2011 ) .
however , the finding that bone resorption on the endocortical surface of the conditional gja1 knockout mice is enhanced under physiological conditions makes the evaluation difficult .
two groups have reported the response to mechanical stress in gja1 conditional knockout mice , in which gja1 is deleted in mature osteoblasts and osteocytes using human osteocalcin promoter cre transgenic mice .
2011 ) have shown that periosteal bone formation is enhanced by mechanical loading in the conditional gja1 knockout mice but not in wild - type mice , whereas lloyd et al .
( 2012 ) have demonstrated that bone formation on both endocortical and periosteal surfaces is decreased in wild - type mice but not in gja1 conditional knockout mice at unloading . as hemichannels and gap junctions ,
both of which are formed by gja1 , play important roles in the response to mechanical stress in osteocytes in vitro ( cherian et al .
2005 ; batra et al . 2012 ) , we need to evaluate the physiological importance of gja1 in the response to mechanical stress by using osteocyte - specific gja1 conditional knockout mice . in the unloaded condition , bone resorption is enhanced and bone formation is mildly suppressed in trabecular bone of wild - type mice but not of bcl2 transgenic mice at 4  months of age , indicating that the osteocyte network is responsible for bone loss at unloading ( moriishi et al .
rankl is upregulated in osteoblasts of wild - type mice but not of bcl2 transgenic mice at unloading ( moriishi et al . 2012 ) . therefore
, the osteocyte network is required for the induction of rankl in osteoblasts at unloading .
although rankl upregulation in osteocytes in the unloaded condition might increase bone resorption by reducing opg release from osteocytes to the bone surface , the upregulation of rankl in osteocytes by unloading is inconsistently observed ( xiong et al .
the reduction in bone formation in response to unloading is abrogated in sost mice and the level of sost mrna but not sost - positive osteocytes is increased after unloading ( robling et al .
further , the downregulation of sost after loading is dependent on the site in the tibiae ( moustafa et al .
, sost - positive cells are regionally increased in wild - type mice but not in bcl2 transgenic mice at 4  months of age ( moriishi et al .
therefore , the osteocyte network is required for the upregulation of sost expression at unloading , which is , at least in part , responsible for the reduction of bone formation in the unloaded condition .
many other molecules , including pth , estrogen receptor  , androgen receptor , leptin , periostin , 1 integrin and il-11 , are also involved in the response to mechanical loading ( price et al .
loading above physiological conditions activates anabolic pathways and suppresses anti - anabolic pathways for bone formation and makes the overall function of osteocytes more anabolic .
recently , we have shown that the mrna expression of pyruvate dehydrogenase kinase 4 ( pdk4 ) , which inactivates the pyruvate dehydrogenase complex , is upregulated in osteoblasts and osteocytes in the unloaded condition in wild - type mice but not in bcl2 transgenic mice at 4  months of age and that bone resorption is not enhanced in the unloaded condition in pdk4-deficient mice .
thus , pdk4 is involved in a catabolic pathway in the unloaded condition ( wang et al .
gene targeting experiments , especially the generation of osteocyte - specific conditional knockout mice , are revealing the importance of osteocytes in bone modeling and remodeling .
although these experiments have uncovered the function of individual genes in osteocytes , the overall function of the osteocyte network remains to be clarified , because osteocyte death causes apoptosis- or necrosis - induced bone resorption .
indeed , an ideal mouse model for the evaluation of the overall function of the osteocyte network seems to be an impossibility .
taking into consideration recent findings from osteocyte - network - disrupted mouse models with previous histological observations , however , the overall functions of the osteocyte network are now evident .
the osteocyte network enhances bone resorption by activating osteoclastogenesis but inhibits bone formation by suppressing osteoblast function under physiological conditions . in the unloaded condition ,
the functions of the osteocyte network are augmented and bone resorption is further enhanced and bone formation is further inhibited .
these functions of the osteocyte network also explain the increases in bone mass seen after exercise in many clinical studies ( schwab and klein 2008 ) , because the osteocyte network decreases the inhibitory effects on bone mass by reducing the stimulatory effect on osteoclastogenesis and the inhibitory effect on osteoblast function in the loaded condition ( under physiological conditions ) compared with the unloaded condition .
 osteocytes establish an extensive intracellular and extracellular communication system via gap - junction - coupled cell processes and canaliculi throughout bone and the communication system is extended to osteoblasts on the bone surface . 
 the osteocyte network is an ideal mechanosensory system and suitable for mechanotransduction . 
 however , the overall function of the osteocyte network remains to be clarified , since bone resorption is enhanced by osteocyte apoptosis , which is followed by a process of secondary necrosis attributable to the lack of scavengers . 
 the enhanced bone resorption is caused by the release of intracellular content , including immunostimulatory molecules that activate osteoclastogenesis through the canaliculi . 
 therefore , a mouse model is required in which the osteocyte network is disrupted but in which no bone resorption is induced , in order to evaluate the overall functions of the osteocyte network . 
 one such model is the bcl2 transgenic mouse , in which the osteocyte network , including both intracellular and extracellular networks , is disrupted . 
 another model is the osteocyte - specific gja1 knockout mouse , in which intercellular communication through gap junctions is impaired but the canalicular system is intact . combining the findings from these mouse models with previous histological observations showing the inverse linkage between osteocyte density and bone formation , 
 we conclude that the osteocyte network enhances bone resorption and inhibits bone formation under physiological conditions . 
 further , studies with bcl2 transgenic mice show that these osteocyte functions are augmented in the unloaded condition . in this condition , rankl upregulation in osteoblasts and sost upregulation in osteocytes 
 are , at least in part , responsible for enhanced bone resorption and suppressed bone formation , respectively . 
Introduction
Osteocyte death and bone remodeling
Functions of osteocytes in bone resorption
Functions of osteocytes in bone formation
Regulation of bone mass by mechanical stress
Concluding remarks