bone formation , also known as osteogenesis , is a complicated process that underlies the whole life of a body . the process of osteogenesis is comprised of maturation of osteoblasts and osteocytes , extracellular excretion , and ossification . osteoblasts are the most important cells in bone tissues which are critical for bone formation . ossification has a restrict order : first , collagen proteins accumulate in immature bone tissue ; second , osteoblasts secrete alkaline phosphatase ( alp ) , osteopontin ( opn ) , and osteocalcin ( ocn ) ; and third , bone canaliculus forms finally . bone mesenchymal stem cells ( bmscs ) are precursors of osteoblasts , which can differentiate into chondrocytes , adipocytes , and osteoblasts . the most cellular events involved in bone formation include the proliferation and differentiation of bmscs . many studies have revealed that bone morphogenetic proteins ( bmps ) , belong to the transforming growth factor beta superfamily , stimulate proliferation and differentiation of bmscs and enhance mineralization of skeleton tissue . it is also known that wnt proteins signals which regulate cell growth , differentiation , function , and death , are essential for bone mass regulation . prostaglandin ( pg ) , mainly produced by cyclooxygenase-2 ( cox-2 ) , is reported with the significant role of bone formation by forming an important regulatory loop with bmps . moreover , glucocorticoids ( gcs ) exert ambiguous effects to bmscs and osteoblasts , low dose of gcs keeps hemostasis of bone tissues while high dose inhibits bone formation . recently , several researchers revealed that epithelium sodium channel ( enac ) may be the importance of regulation of bmscs and osteoblasts . the amiloride - sensitive enacs , first described in 1994 , is a membrane - bound ion - channel that is permeable for li - ions , protons , and especially sodium ions . meanwhile , this sodium channels are characterized primarily by their high affinity to the diuretic blocker amiloride . studies showed that enacs were abundantly expressed in the tissues of kidney , colon , lung , sweat glands , and reproductive ducts . furthermore , recent studies have discovered that enac may also be expressed in osteoblasts and mscs , for sodium concentration in the extracellular fluid may affect proliferation and differentiation of osteoblasts . therefore , the study on enacs may provide a new sight to understand the process and regulation of bone formation . in this review , we retrospect researches within the last 5 years and try to illustrate the characteristics and regulation of enac as well as the function of enac during bone formation . enacs are members of the enac / degenerin family of ion channels , which are assembled from homologous  ,  , and  subunits . each subunit consists of two transmembrane helices and an extracellular loop , and shares a similar secondary structure consisting of a large extracellular region linked to two membrane spanning domains ( tm1 and tm2 ) , and short intracellular n- and c - termini . all three subunits are essential for transporting to the membrane assembly of functional channels on the membrane . enac highly seems to be a heterotrimeric protein like the recently analyzed acid - sensing ion - channel 1 ( asic1 ) , which belongs to the same family . crystal structure of enacs and site - directed mutagenesis studies suggest that enac has a central ion - channel located on the central symmetry axis of the three subunits . they are made of an intracellular n - terminus region followed by a transmembrane domain , a large extracellular loop , a second transmembrane segment , and a c - terminal intracellular tail . the extracellular region of asic1 has a highly ordered structure containing domains formed either by  strands or  helices . domains within the structure are aptly named wrist , finger , thumb , palm , knuckle , and -ball [ figure 1 ] . the structure of antiracism study - dialogue circle 1 is similar to an outstretched hand containing a ball .. enacs are protease sensitive channels whose iron - channel activity is regulated by the proteolytic reaction . trypsin and chymotrypsin activate enacs in the epithelium of endometrium of the uterus to help fertilization of oocytes . contrastly , extracellular serine protease inhibitors , such as aprotinin and bikunin , have been shown to decrease channel activity . in addition , furin , a proprotein convertase that cleaves latent precursor proteins into biologically active products , cleaves  and  subunits of enacs during its maturation . the  subunit is cleaved by furin only once and release 18,000 and 75,000 fragments of proteolytic peptides [ figure 2 ] . remaining peptides after treatment of furin to  and  subunits of epithelium sodium channels . furin cleaves  subunit and releases 65,000 and 30,000 peptides and a 26 residue fragment . channel opening probability ( po ) of enacs is regulated by several physical and chemical factors . as mentioned , enacs are proteinase sensitive iron - channel , proteinase - treated enacs performing a high rate po . notably , the release of an inhibitory tract activates the channel by increasing its open po , meanwhile , subunits must be cleaved at least twice at sites flanking the inhibitory tract to be activated . furin cleaves  subunit twice releasing a 65,000 , a 30,000 peptide and a 26 residue fragment , enhance permeable of cations . however , as furin cleaves the  subunit once , a second protease cleaving the  subunit could release the inhibitory tract and improve iron permeability . moreover , trypsin which is a serine protease activating enac induces an inward whole - cell current and the augment of the current could be abolished by amiloride , an enac inhibitor or by aprotinin . in addition , renal tubule epithelia , especially apical membrane , which are regulated by aldosterone or tubular volume . prostasin is observed to have a role in  subunit under the regulation of volume depletion and aldosterone excretion . another factor that affects po of enacs is a mechanical force . studying enacs of caenorhabditis elegans reveals a fact that enacs are members of a family of ion channels that own a character of mechanical - sensitive . the negative pressure of collecting ducts which is up to 80 mmhg elicits an increase in channel po . an increased volume of the bladder , resulting in an augment of the hydrostatic pressure of endothelia of bladder , could induce improved inward whole - cell current which is partly abolished by amiloride . renal tubule epithelia are exposed under the flow of urine which creates a condition of flow stress . it is still not unclear whether the enhanced effect of shear stress could be abrogated by amiloride , as enacs are abundantly expressed on osteoblasts . however , enac might play a significant role in shear stress further regulated osteoblast maturation . enacs are members of the enac / degenerin family of ion channels , which are assembled from homologous  ,  , and  subunits . each subunit consists of two transmembrane helices and an extracellular loop , and shares a similar secondary structure consisting of a large extracellular region linked to two membrane spanning domains ( tm1 and tm2 ) , and short intracellular n- and c - termini . all three subunits are essential for transporting to the membrane assembly of functional channels on the membrane . enac highly seems to be a heterotrimeric protein like the recently analyzed acid - sensing ion - channel 1 ( asic1 ) , which belongs to the same family . crystal structure of enacs and site - directed mutagenesis studies suggest that enac has a central ion - channel located on the central symmetry axis of the three subunits . they are made of an intracellular n - terminus region followed by a transmembrane domain , a large extracellular loop , a second transmembrane segment , and a c - terminal intracellular tail . the extracellular region of asic1 has a highly ordered structure containing domains formed either by  strands or  helices . domains within the structure are aptly named wrist , finger , thumb , palm , knuckle , and -ball [ figure 1 ] . the structure of antiracism study - dialogue circle 1 is similar to an outstretched hand containing a ball .. enacs are protease sensitive channels whose iron - channel activity is regulated by the proteolytic reaction . trypsin and chymotrypsin activate enacs in the epithelium of endometrium of the uterus to help fertilization of oocytes . contrastly , extracellular serine protease inhibitors , such as aprotinin and bikunin , have been shown to decrease channel activity . in addition , furin , a proprotein convertase that cleaves latent precursor proteins into biologically active products , cleaves  and  subunits of enacs during its maturation . the  subunit is cleaved by furin only once and release 18,000 and 75,000 fragments of proteolytic peptides [ figure 2 ] . remaining peptides after treatment of furin to  and  subunits of epithelium sodium channels . furin cleaves  subunit and releases 65,000 and 30,000 peptides and a 26 residue fragment . channel opening probability ( po ) of enacs is regulated by several physical and chemical factors . as mentioned , enacs are proteinase sensitive iron - channel , proteinase - treated enacs performing a high rate po . notably , the release of an inhibitory tract activates the channel by increasing its open po , meanwhile , subunits must be cleaved at least twice at sites flanking the inhibitory tract to be activated . furin cleaves  subunit twice releasing a 65,000 , a 30,000 peptide and a 26 residue fragment , enhance permeable of cations . however , as furin cleaves the  subunit once , a second protease cleaving the  subunit could release the inhibitory tract and improve iron permeability . moreover , trypsin which is a serine protease activating enac induces an inward whole - cell current and the augment of the current could be abolished by amiloride , an enac inhibitor or by aprotinin . in addition , renal tubule epithelia , especially apical membrane , which are regulated by aldosterone or tubular volume . prostasin is observed to have a role in  subunit under the regulation of volume depletion and aldosterone excretion . another factor that affects po of enacs is a mechanical force . studying enacs of caenorhabditis elegans reveals a fact that enacs are members of a family of ion channels that own a character of mechanical - sensitive . the negative pressure of collecting ducts which is up to 80 mmhg elicits an increase in channel po . an increased volume of the bladder , resulting in an augment of the hydrostatic pressure of endothelia of bladder , could induce improved inward whole - cell current which is partly abolished by amiloride . renal tubule epithelia are exposed under the flow of urine which creates a condition of flow stress . it is still not unclear whether the enhanced effect of shear stress could be abrogated by amiloride , as enacs are abundantly expressed on osteoblasts . however , enac might play a significant role in shear stress further regulated osteoblast maturation . enacs are critical in proliferation and differentiation of osteoblasts because enacs activate the expression of runt - related transcription factor 2 ( runx2 ) , a key transcriptional modulator of osteoblasts formation which plays a fundamental role in osteoblasts maturation and homeostasis . furthermore , runx2 controls transcription of ossify - specific genes including ocn , opn , and collagenase-3 in bmscs and osteoblasts . as osteoblasts are imbedded in periosteum and metaphysis of bones , different with reproductive tracts , the stimulation of enacs can not be performed by protease release . recent studies have revealed that sensitization of enac in osteoblasts is mainly caused by cyclic guanosine monophosphate ( cgmp ) . exogenous 8-pcpt - cgmp , a cell - permeable cgmp analog , is reported to stimulate the expression of runx2 via increased expression of enacs . consequently , the effect of stimulation could be abolished by either amiloride or small interfering rna ( sirna ) of enac . furthermore , the effect of cgmp on enacs is due to sensitization of cgmp - dependent protein kinase ii ( pkg ii ) and knockdown of pkg ii ( via sirna ) blocked 8-pcpt - cgmp induced expression of enac . although 8-pcpt - cgmp stimulate expression of enac  an augment of enacs ( see below ) , it is still under - investigated whether cgmp could improve the opening po enac . nitride oxide synthases ( nos ) , which synthesis nitride oxide ( no ) , are a series of proteins expressed widely on nearly all cells . it has been revealed that nos has three isoforms , neuronal nos ( nnos ) , endothelial nos ( enos ) , and inducible nos ( inos ) . it also shown that anabolic effect of estrogen is partly mediated by enos isoform , as deletion of the enos leads to an osteoblast - driven mild osteoporotic bone , and finally shows a blunted response to estrogen . inos - derived no also stimulates fracture healing as well as recovery of bone mass . no derived from nnos negatively regulates osteogenesis since nnos knocked - out osteoblasts show an increased level of alp . no derived from nos triggers pkg ii by cgmp which is synthesized by soluble guanylate cyclase ( sgc ) and , therefore , up - regulates the expression of enac . it is also reported that fluid shear stress increased osteoblast no synthesis , leading to activation of pkg ii . this means enac may partly mediate the influence of shear stress and the pkg ii and enac might have a regulation loop in shear stress which activates osteoblast proliferation and differentiation . as discussed above , the activation of enacs triggers influx of sodium iron leading to a consequence that membrane potential is elevated temporarily . the underlying mechanism of elevating potential is velocity and quantity of influx sodium irons pass through opening enacs . importantly , for osteoblasts and oocytes , which are different from duct cells , two factors are related to depolarization of cell membrane  number ( n ) of enacs localized on membrane and activity ( po ) of an individual enac . the result of patch clamps shows that potential of oocytes is declined by 22.24 mv and potential of smc - c6 cells , an immoral epithelium cell line , is dropped by 12.1 mv , both boosting potential abrogated by amiloride . these results consequently suggest that the summarized effects of both n and po of enac in different cells are diverged . voltage - sensitive calcium channels ( vsccs ) respond to the elevated potential through enac activity . in details , serine protease - induced , enac - mediated membrane depolarization could result in ca mobilization . moreover then , the ca level rise was largely lessened in ca - free solutions , while nifedipine , a blocker of the vscc , abolished the trypsin - induced ca level rise . as investigated , vsccs are classified into two types , t - type and l - type vsccs . osteoblasts predominantly express l - type vgccs , whereas osteocytes in mature bone tissues which are derived from osteoblasts , express t - type vsccs and a small amount of l - type . both types of cells in calcium - free medium showed no response to fluid flow . for example , amlodipine , an l - type vscc blocker , is used to treat mc3t3-e1 ( immoral osteoblast cell line ) and mlo - y4 ( immoral osteocyte cell line ) cells showing a prolonged declining of calcium in both types of cells , while treatment of nnc55 - 0396 ( t - type vscc blocker ) can discontinue the repetitive influx calcium in mlo - y4 cells after inducing an immediate spike , and it had no observable effects on mc3t3-e1 cells . it is reported that influx calcium irons resulted in physiology change of osteoblasts . in details , increased intracellular calcium leads to activation of constitutively expressed no synthases , and increased no production in osteoblasts . no further activates pkg ii , which phosphorylates substrates make the mek / erk pathway activated . furthermore , the mek / erk pathway increases transcription of c - fos , fra-1 , fra-2 , and fosb , which is via the following activation of camp - response element binding protein ( creb ) , direct phosphorylation of elk and recruitment of ap-1 complexes . these fos family genes encode members of the ap-1 transcription factor family that regulate osteoblast proliferation and differentiation . for another , elevated intracellular calcium has been reported to facilitate cox2 induced production of pge2 and calcium promotes cox transcription via activating of creb . pge2 derived from cox2 enhances the expression of alp , an indicator of bone formation , and could be impeded by ru486 , an inhibitor of cox2 . therefore , the absence of cox2 would be expected to decrease both bone formation and bone resorption . moreover , cox2 may form an important regulatory loop with bmp / smad family in bmscs and osteoblasts and further have an influence on runx2 , a key transcriptional modulator in bone formation . transmembrane potential is decreased leading to stimulation of voltage - sensitive calcium channel accompanying with calcium influx . for one thing , nitride oxide activates soluble guanylate cyclase and the resulting cyclic guanosine monophosphate activates cyclic guanosine monophosphate - dependent protein kinase ii , which phosphorylates substrates leading to activation of the mek / erk pathway . extracellular nitride oxide from endothelium e.g. , may also stimulate mek / erk pathway . for another , activated cyclic guanosine monophosphate - dependent protein kinase ii may also enhance expression of epithelium sodium channels . pge2 derived from cyclooxygenase-2 forms a regulation loop with bone morphogenetic protein / smad family . recent studies have revealed that sensitization of enac in osteoblasts is mainly caused by cyclic guanosine monophosphate ( cgmp ) . exogenous 8-pcpt - cgmp , a cell - permeable cgmp analog , is reported to stimulate the expression of runx2 via increased expression of enacs . consequently , the effect of stimulation could be abolished by either amiloride or small interfering rna ( sirna ) of enac . furthermore , the effect of cgmp on enacs is due to sensitization of cgmp - dependent protein kinase ii ( pkg ii ) and knockdown of pkg ii ( via sirna ) blocked 8-pcpt - cgmp induced expression of enac . although 8-pcpt - cgmp stimulate expression of enac  an augment of enacs ( see below ) , it is still under - investigated whether cgmp could improve the opening po enac . nitride oxide synthases ( nos ) , which synthesis nitride oxide ( no ) , are a series of proteins expressed widely on nearly all cells . it has been revealed that nos has three isoforms , neuronal nos ( nnos ) , endothelial nos ( enos ) , and inducible nos ( inos ) . it also shown that anabolic effect of estrogen is partly mediated by enos isoform , as deletion of the enos leads to an osteoblast - driven mild osteoporotic bone , and finally shows a blunted response to estrogen . moreover , inos - derived no also stimulates fracture healing as well as recovery of bone mass . no derived from nnos negatively regulates osteogenesis since nnos knocked - out osteoblasts show an increased level of alp . no derived from nos triggers pkg ii by cgmp which is synthesized by soluble guanylate cyclase ( sgc ) and , therefore , up - regulates the expression of enac . it is also reported that fluid shear stress increased osteoblast no synthesis , leading to activation of pkg ii . this means enac may partly mediate the influence of shear stress and the pkg ii and enac might have a regulation loop in shear stress which activates osteoblast proliferation and differentiation . as discussed above , the activation of enacs triggers influx of sodium iron leading to a consequence that membrane potential is elevated temporarily . the underlying mechanism of elevating potential is velocity and quantity of influx sodium irons pass through opening enacs . importantly , for osteoblasts and oocytes , which are different from duct cells , two factors are related to depolarization of cell membrane  number ( n ) of enacs localized on membrane and activity ( po ) of an individual enac . the result of patch clamps shows that potential of oocytes is declined by 22.24 mv and potential of smc - c6 cells , an immoral epithelium cell line , is dropped by 12.1 mv , both boosting potential abrogated by amiloride . these results consequently suggest that the summarized effects of both n and po of enac in different cells are diverged . voltage - sensitive calcium channels ( vsccs ) respond to the elevated potential through enac activity . in details , serine protease - induced , enac - mediated membrane depolarization could result in ca mobilization . moreover then , the ca level rise was largely lessened in ca - free solutions , while nifedipine , a blocker of the vscc , abolished the trypsin - induced ca level rise . as investigated , vsccs are classified into two types , t - type and l - type vsccs . osteoblasts predominantly express l - type vgccs , whereas osteocytes in mature bone tissues which are derived from osteoblasts , express t - type vsccs and a small amount of l - type . both types of cells in calcium - free medium showed no response to fluid flow . for example , amlodipine , an l - type vscc blocker , is used to treat mc3t3-e1 ( immoral osteoblast cell line ) and mlo - y4 ( immoral osteocyte cell line ) cells showing a prolonged declining of calcium in both types of cells , while treatment of nnc55 - 0396 ( t - type vscc blocker ) can discontinue the repetitive influx calcium in mlo - y4 cells after inducing an immediate spike , and it had no observable effects on mc3t3-e1 cells . it is reported that influx calcium irons resulted in physiology change of osteoblasts . in details , increased intracellular calcium leads to activation of constitutively expressed no synthases , and increased no production in osteoblasts . no further activates pkg ii , which phosphorylates substrates make the mek / erk pathway activated . furthermore , the mek / erk pathway increases transcription of c - fos , fra-1 , fra-2 , and fosb , which is via the following activation of camp - response element binding protein ( creb ) , direct phosphorylation of elk and recruitment of ap-1 complexes . these fos family genes encode members of the ap-1 transcription factor family that regulate osteoblast proliferation and differentiation . for another , elevated intracellular calcium has been reported to facilitate cox2 induced production of pge2 and calcium promotes cox transcription via activating of creb . pge2 derived from cox2 enhances the expression of alp , an indicator of bone formation , and could be impeded by ru486 , an inhibitor of cox2 . therefore , the absence of cox2 would be expected to decrease both bone formation and bone resorption . moreover , cox2 may form an important regulatory loop with bmp / smad family in bmscs and osteoblasts and further have an influence on runx2 , a key transcriptional modulator in bone formation . transmembrane potential is decreased leading to stimulation of voltage - sensitive calcium channel accompanying with calcium influx . for one thing , nitride oxide activates soluble guanylate cyclase and the resulting cyclic guanosine monophosphate activates cyclic guanosine monophosphate - dependent protein kinase ii , which phosphorylates substrates leading to activation of the mek / erk pathway . extracellular nitride oxide from endothelium e.g. , may also stimulate mek / erk pathway . for another , activated cyclic guanosine monophosphate - dependent protein kinase ii may also enhance expression of epithelium sodium channels . pge2 derived from cyclooxygenase-2 forms a regulation loop with bone morphogenetic protein / smad family . although enacs are regulated by angiotensin , vasopressin , and aldosterone in various organs , it is still unclear whether enacs expression in osteoblasts is related to these hormone . interestingly , estrogen regulates bmscs , osteoblasts and osteocytes via several different signal pathways , mainly by wnt and rankl signals . these may probably answer the question why estrogen protects premenopausal women . however , it has been investigated that estrogen has maintained bone density via enacs because estrogen regulates the expression of enac  and  subunits in osteoblasts . patch clamps result shows that enac activity changes and increases influx current of whole cells as estrogen is added to the medium . therefore , both results suggest that estrogen not only improves po of enac but also increases no of enacs in an individual cell . in addition , research of tang et al . presents a consistent result that raloxifene , the selective estrogen receptor modulator , collaborates with the effect of shear stress on enacs , enhancing proliferation of osteoblasts . further studies focusing on the mechanism of estrogen on enacs show that no may act as a mediator of estrogen role in bone formation . for example , mice with nos deficiency lost bone normally following ovariectomy but exhibited a significantly blunted anabolic response to high - dose exogenous estrogen . however , osteoblasts with nos knocked - out had reduced rates of growth . as discussed above , no derived up - regulates the expression of enac via the cgmp - pkg pathway , the number of enacs on an individual osteoblast is increased by estrogen partly via no synthesis . although we partly illustrate the role of enacs on bone formation , the effect and regulations mechanism of enacs are still not quite clear . the main doubt is whether enacs , which are regulated via cgmp / pkg ii pathway , can stimulate the synthesis of cgmp via nos stimulation . as a fact , the regulation of enac is not out of control ; thus , more mechanisms to be unveiled may participate in the regulation of enacs . furthermore , it is still not well known about the pathways that estrogen participates in . last but not the least , high - salt dietary may cause osteoporosis and the hypothesis raised is that the calciuria is partly due to salt - induced volume expansion which causes excretion of parathyroid hormone leading to bone remodeling . in fact , the adult human body contains 90130 g sodium , roughly half of that stored in bone . as fluctuate of plasma and intracellular sodium may affect the function ( po ) of enacs , a bold suspicion is whether high - salt dietary affects bone remodeling via enacs expressed on the surface of osteoblasts and osteocytes . this work was supported by a grant of the national natural science foundation of china ( no . this work was supported by a grant of the national natural science foundation of china ( no .
objective : to review the recent developments in the mechanisms of epithelium sodium channels ( enacs ) induced bone formation and regulation.data sources : studies written in english or chinese were searched using medline , pubmed and the index of chinese - language literature with time restriction from 2005 to 2014 . 
 keywords included enac , bone , bone formation , osteonecrosis , estrogen , and osteoporosis . 
 data from published articles about the structure of enac , mechanism of enac in bone formation in recent domestic and foreign literature were selected.study selection : abstract and full text of all studies were required to obtain . 
 studies those were not accessible and those did not focus on the keywords were excluded.results:enacs are tripolymer ion channels which are assembled from homologous  ,  , and  subunits . 
 crystal structure of enacs suggests that enac has a central ion - channel located in the central symmetry axis of the three subunits . 
 enacs are protease sensitive channels whose iron - channel activity is regulated by the proteolytic reaction . 
 channel opening probability of enacs is regulated by proteinases , mechanical force , and shear stress . 
 several molecules are involved in regulation of enacs in bone formation , including nitride oxide synthases , voltage - sensitive calcium channels , and cyclooxygenase-2.conclusion:the pathway of enac involved in shear stress has an effect on stimulating osteoblasts even bone formation by estrogen interference .