Molecular mechanisms involved in hypoxia-induced alterations in bone remodeling

  Ci a ion: Usa egui-Ma ín, R.; Rigual, R.; Ruiz-Mamb illa, M.; Fe nández- Gómez, J.-M.; Dueñas, A.; Pé ez- Cas illón, J.L. Molecula Mechanisms In ol ed in Hypoxia-Induced Al e a ions in Bone Remodeling. In . J. Mol. Sci. 2022,23, 3233. h ps://doi.o g/10.3390/ ijms23063233 Academic Edi o : Giacomina B une i Recei ed: 25 Feb ua y 2022 Accep ed: 15 Ma ch 2022 Published: 17 Ma ch 2022 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2022 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). In e na ional Jou nal o Molecula Sciences Re iew Molecula Mechanisms In ol ed in Hypoxia-Induced Al e a ions in Bone Remodeling Rica do Usa egui-Ma ín1,2,*, Rica do Rigual 3,4, Ma a Ruiz-Mamb illa 5, José-Ma ía Fe nández-Gómez 1, An onio Dueñas 6,7 and JoséLuis Pé ez-Cas illón6,8,* 1Depa men o Cell Biology, His ology and Pha macology, Facul y o Medicine, Uni e si y o Valladolid, 47003 Valladolid, Spain; [email p o ec ed] 2IOBA, Uni e si y o Valladolid, 47011 Valladolid, Spain 3 Depa men o Biochemis y, Molecula Biology and Physiology, Facul y o Medicine, Uni e si y o Valladolid, 47003 Valladolid, Spain; [email p o ec ed] 4IBGM, Uni e si y o Valladolid, 47003 Valladolid, Spain 5Depa men o Su ge y, Facul y o Medicine, Uni e si y o Valladolid, 47003 Valladolid, Spain; [email p o ec ed] 6Depa men o Medicine, Facul y o Medicine, Uni e si y o Valladolid, 47003 Valladolid, Spain; [email p o ec ed] 7Depa men o Toxicology, Río Ho ega Uni e si y Hospi al, 47012 Valladolid, Spain 8Depa men o In e nal Medicine, Río Ho ega Uni e si y Hospi al, 47012 Valladolid, Spain *Co espondence: [email p o ec ed] (R.U.-M.); [email p o ec ed] (J.L.P.-C.) Abs ac : Bone is c ucial o he suppo o muscles and he p o ec ion o i al o gans, and as a ese oi o calcium and phospho us. Bone is one o he mos me abolically ac i e issues and is con inuously enewed o adap o he changes equi ed o heal hy unc ioning. To main ain no mal cellula and physiological bone unc ions su icien oxygen is equi ed, as e idence has shown ha hypoxia may in luence bone heal h. In his scena io, his e iew aimed o analyze he molecula mechanisms in ol ed in hypoxia-induced bone emodeling al e a ions and hei possible clinical consequences. Hypoxia has been associa ed wi h educed bone o ma ion and educed os eoblas ma ix mine aliza ion due o he hypoxia en i onmen inhibi ing os eoblas di e en ia ion. A hypoxic en i onmen is in ol ed wi h inc eased os eoclas ogenesis and inc eased bone eso p i e capaci y o he os eoclas s. Clinical s udies, al hough wi h con adic o y esul s, ha e shown ha hypoxia can modi y bone emodeling. Keywo ds: hypoxia; bone emodeling; HIF; os eoclas ; os eoblas ; oxygen 1. In oduc ion Bone is one o he mos me abolically ac i e issues and is con inuously enewed o keep he skele on heal hy. This con inuous emodeling allows bone o adap o he changes equi ed o heal hy unc ioning and main ain bone s eng h. Bone has h ee undamen al unc ions: suppo o he muscles, he p o ec ion o i al o gans, and as a ese oi o cal- cium and phospho us o ca y ou a ious me abolic unc ions [ 1 ]. Os eoblas s, os eoclas s, and os eocy es a e he p incipal cell componen s o he bone issue. Bone ex acellula ma ix has wo componen s, a mine al pa comp ising hyd oxyapa i e ( 70–90% ) and an o ganic pa o p ima ily collagen (app ox. 90%) and non-collagenous p o eins such as sialop o ein, os eonec in, os eopon in, and os eocalcin (app ox. 10%) [ 1 – 3 ]. In ma u e bone, wo his ological ypes can be iden i ied: co ical and abecula bone. Co ical bone is compac , dense, o de ed bone and ep esen s 80% o o al bone mass. T abecula bone is ligh e and less compac , wi h an i egula s uc u e [1,4,5]. Su icien oxygen is equi ed o main ain op imal unc ioning o cells and issues. In he absence o su icien oxygen, unde hypoxic condi ions, cells mus make molecula and physiological adjus men s o p olong hei su i al [6]. In his scena io, e idence has In . J. Mol. Sci. 2022,23, 3233. h ps://doi.o g/10.3390/ijms23063233 h ps://www.mdpi.com/jou nal/ijms In . J. Mol. Sci. 2022,23, 3233 2 o 12 shown ha hypoxia may in luence bone heal h [7–9]. Thus, his e iew aimed o examine he molecula mechanisms in ol ed in hypoxia-induced al e a ions in bone emodeling. We made a comp ehensi e e iew o he li e a u e h ough MEDLINE, PubMed, Web o Science, Scopus, and Embase elec onic da abases. Po en ially ele an a icles we e sough by using he sea ch e ms in combina ion as Medical Subjec Headings (MeSH) e ms and ex wo ds: “bone”, “bone emodeling”, “hypoxia”, “bone me abolism”, “os eoclas ”, “os eoblas ” and “os eocy e”. In addi ion, we scanned he e e ence lis s o he e ie ed publica ions o iden i y addi ional ele an a icles. The sea ch was supplemen ed using he MedLine op ion ‘Rela ed A icles’. No language es ic ions we e applied. The abs ac s o each a icle we e s udied o ensu e ele ance and signi icance. 2. Bone Remodeling and Cells In ol ed The pu pose o bone emodeling is o main ain bone biomechanical s abili y and is ca ied ou in an ana omical and unc ional s uc u e called a bone emodeling uni (BMU). BMU in ol es ou ypes o cells: bone lining cells, os eocy es, os eoclas s, and os eoblas s. The lining cells, om an os eoblas ic lineage, co e he bone su ace du ing he s able phase o emodeling. The mos nume ous bone cells a e he os eocy es, which ac mechanis ically by de ec ing a eas o weakness in he skele on and sending signals o ac i a e emodeling. Os eocy es a e di e en ia ed os eoblas s encased in he bone ma ix. The os eoclas s a e mul inuclea ed cells ha ca y ou bone eso p ion and communica e wi h he os eoblas s o egula e bone o ma ion [10]. The emodeling cycle is 120 days and in ol es he emo al o mine alized bone by os eoclas s, ollowed by he o ma ion o bone ma ix by os eoblas s ha subsequen ly min- e alize. The bone emodeling cycle consis s o he ollowing phases: ini ia ion/ac i a ion phase o si e-speci ic emodeling; eso p ion phase, du ing which os eoclas s diges bone; e e sal phase, in which mesenchymal s em cells and os eoblas p ogeni o s a e ec ui ed on he bone su ace; he o ma ion phase, in which os eoblas s a e ac i a ed and lay down new bone; and he mine aliza ion phase [ 11 ]. In he i s h ee weeks, os eoclas -media ed bone eso p ion and subsequen os eoclas –os eoblas coupling occu . The bone emodeling cycle is egula ed by sys emic and local ac o s. The p incipal sys emic ac o s a e pa a hy oid ho mone (PTH), g ow h ho mone, calci iol, hy oid ho mones, glucoco icoids, and sex ho mones. In addi ion, egula o s such as insulin-like g ow h ac o s (IGFs), umo g ow h ac o -be a (TGF- β ), bone mo phogene ic p o eins (BMP), p os aglandins, and cy okines a e also in ol ed. A la ge numbe o cy okines and g ow h ac o s a ec bone cell unc ions and ac as local egula o s. The ecep o ac i a o o he NF- κ B (RANK)- ecep o ac i a o o he NF- κ B ligand (RANKL)-os eop o ege in (OPG) pa hway also is c ucial in bone emodeling egula ion [11,12]. 2.1. Os eocy es The os eocy es a e cells di e en ia ed om os eoblas s ha a e included wi hin he bone ma ix in such a way ha app oxima ely 5–20% o os eoblas s a e ans o med in o os eocy es. The p ecise mechanism and he molecula and gene ic egula ion by which an os eoblas di e en ia es in o an os eocy e ha embeds in o he bone ma ix a e no ye ully unde s ood. I has been hypo hesized o be a passi e p ocess in which a subpopula ion o os eoblas s s op hei syn hesis o bone ma ix and become bu ied cells benea h he bone ma ix syn hesized by o he os eoblas s. On he o he hand, ano he hypo hesis is ha i may be an ac i e and in asi e p ocess in which is equi ed a ma ix deg ada ion o he o ma ion o he os eocy e lacuna [ 13 ]. These cells exp ess simila genes o os eoblas s, being pa icula ly impo an in he exp ession o genes in ol ed in bone mine aliza ion and phospho us me abolism. Os eocy es embedded in he mine alized bone ma ix a e mul i unc ional cells wi h many key egula o y oles in bone emodeling and mine al homeos asis. Os eocy es con ol bone me abolism by egula ing os eoclas s and os eoblas s and a e mechanosenso y cells ha egula e and coo dina e skele al esponses. Os eocy es In . J. Mol. Sci. 2022,23, 3233 3 o 12 play a c ucial ole in bone calcium deposi ion and se e as endoc ine cells ha a e impo an in he con ol o phospha e homeos asis [13,14]. Os eocy es a e he p incipal p oduce s o scle os in (encoded by he SOST gene). Scle os in an agonizes se e al membe s o BMP and binds o LPR5/LPR6, blocking he canonical Wn pa hway inhibi ing he ac i i y o os eoblas s [ 15 ]. In addi ion o blocking bone o ma ion, os eocy es also s imula e he eso p ion p ocess by sec e ing RANKL o s imula ing RANKL p oduc ion by os eoblas s o o he cells o he os eoblas ic lin- eage. Os eocy es also p oduce OPG, which compe es wi h RANKL and hus blocks bone eso p ion [15,16]. 2.2. Os eoclas s Os eoclas s ha e wo main unc ions, bone eso p ion and he ini ia ion o bone o ma ion h ough communica ion wi h os eoblas s. The os eoclas s a e di ec ed o he BMU om he bone ma ow o om os eoclas p ecu so s loca ed in he bloods eam [ 17 ]. In he os eoclas su ace, nume ous ecep o s de e mine hei p oli e a ion, di e en ia ion, and su i al. One o he mos impo an is RANK, he binding si e o RANKL. The exp ession o RANK is s imula ed by he mac ophage colony-s imula o ac o (M-CSF) sec e ed by os eoblas s and bone ma ow s omal cells. The RANK–RANKL in e ac ion ac i a es he NF- κ B pa hway and di e en kinases, whose inal e ec o is he NAFATc1 ac o , which is ansloca ed o he nucleus, ac i a ing he exp ession o genes in ol ed in os eoclas p oli e a ion and ma u a ion. O he impo an ecep o s o TNF α , S c, and TERM2 also coope a e in he ac i a ion o he NAFATc1 ac o [18]. Os eoclas ac i a ion is necessa y o bone eso p ion. Fi s ly, os eoclas s mus adhe e o he bone su ace, ca ied ou by os eoclas podosomes wi h he pa icipa ion o in eg ins, c ea ing he eso p ion gap. Hyd ogen ions a e eleased in he eso p ion gap, which acidi- ies he medium and allows he ac i a ion o hyd oly ic enzymes such as ca hepsin, which deg ades he bone ma ix. The de ached ma e ial (collagen and calcium) is eabso bed by os eoclas s and subsequen ly eleased o he ex e io [ 17 ]. Finally, once he p ocess is comple e, apop osis, he p og ammed cell dea h o os eoclas s, occu s. Howe e , be o e his, os eoblas ac i a ion mus ake place. This occu s in h ee ways. G ow h ac o s embedded in he bone ma ix, called ma icins, a e eleased due o he ac i i y o he os eoclas s, he mos impo an o which a e TGF- β , IGF-1, and BMP2. The os eoblas s a e also ac i a ed by os eoblas –os eoclas con ac h ough ansmemb ane p o eins such as he eph in sys em. Finally, he elease o os eoclas okines, which may be s imula o y o inhibi o y, is also impo an [19]. 2.3. Os eoblas s Os eoblas s a e cell de i ed om mesenchymal p ogeni o s. Di e en popula ions o os eoblas s a e dis inguished; os eoblas p ecu so s om he eso p ion gap a ac ed by subs ances eleased om he bone ma ix, su ace os eoblas s ac i a ed by os eocy es, and os eoblas s bu ied in he bone ma ix. Os eoblas s a e cells wi h a basophilic cy o- plasm, abundan mi ochond ia, and a highly-de eloped Golgi appa a us. This s uc u e explains hei abili y o syn hesize p o eins such as alkaline phospha ase, os eocalcin, and ype I collagen o o m os eoid, which is hen mine alized wi h calcium hyd oxyapa i e c ys als [20,21]. T ansc ip ion ac o s such as Sox9, Runx2, and A -4 play an impo an ole in os- eoblas di e en ia ion and ma u a ion. The ansc ip ion ac o Sox9 is no exp essed in ma u e os eoblas s bu is c ucial in he di e en ia ion om p e-os eoblas s. Runx2 is in ol ed in di e en ia ion and is also impo an in he unc ion o ma u e os eoblas s [ 22 ]. A -4 egula es os eocalcin exp ession, an os eoblas ic p o ein in ol ed in glucose and RANKL egula ion p omo ing os eoblas di e en ia ion and i s unc ion. These ansc ip- ion ac o s a e ac i a ed h ough exogenous ac o s such as TGF- β , IGF-1, and ib oblas g ow h ac o (FGF) [ 19 , 23 ]. Du ing eso p ion, TGF- β is eleased om he bone ma ix, p omo ing he p oli e a ion o os eoblas p ecu so s and he a i al o ma u e os eoblas s In . J. Mol. Sci. 2022,23, 3233 4 o 12 in o he eso p ion gap. TGF- β also s imula es os eoblas p oli e a ion and inc eases he syn hesis o he ma ix. TGF- β inhibi s la e os eoblas ma u a ion and os eocy e apop o- sis, and he e o e is c ucial in he os eoblas /os eoclas a io. IGF-1 and FGF de elop an anabolic unc ion by ac i a ing os eoblas ma u a ion and p omo ing p ecu so chemo- axis [ 19 , 23 , 24 ]. These ac o s ac on ecep o s loca ed on he su ace o he os eoblas , ac i a ing me abolic pa hways which s imula e he ansc ip ion ac o s p e iously de- sc ibed. O pa icula no e a e he No ch pa hway, he Hedgehog signaling pa hway, he Wn pa hway, and he BMP pa hway [ 19 , 25 , 26 ]. In addi ion o gene ic con ol o hese signaling pa hways, epigene ic con ol h ough mic o RNA (miRNA) and small agmen s o o he non-coding RNA (ncRNA) ha egula e pos - ansc ip ional gene exp ession by inhibi ing hei ansla ion o s imula ing hei deg ada ion a e impo an [27]. 3. Hypoxia: Local E ec s on Bone Me abolism Cells Hypoxia is a condi ion in which cell unc ion in limi ed by dep i a ion o adequa e oxygen concen a ion. The cellula hypoxic esponse is ini ia ed by hypoxic s imuli such as low oxygen p essu e, poo oxygen di usion-pe usion, among o he s, and is medi- a ed by hypoxia-inducible ac o s (HIF). HIF consis s o wo subuni s: an HIF- α subuni (oxygen-sensi i e) and a cons i u i ely exp essed uni , HIF- β [ 28 , 29 ]. In no moxia, HIF- α is hyd oxyla ed by p olyl hyd oxylase domain iso o ms (PHDs) and is hen polyubiqui ina ed by he on Hippel-Lindau (VHL) p o ein o p o easomal deg ada ion. In no moxia, ac o - inhibi ing hypoxia (FIH) hyd oxyla es HIF- α , inhibi ing i s ansc ip ional capaci y [ 30 , 31 ]. In hypoxia, he ac i i y o PHD and FIH a e educed, leading o he accumula ion o HIF- α and i s ansloca ion o he nucleus. In he nucleus, he he e odime HIF- α /HIF- β is o med and o ms a complex wi h he cAMP esponse elemen -binding (CREB), he binding p o ein (CBP1), and his one ace yl ans e ase p300. This complex binds o he hypoxia esponse elemen s (HRE) o HIF a ge genes o ac i a e he exp ession o mo e han 200 genes, ini ia ing he s imula ion o se e al cellula pa hways aimed a su i al in a hypoxic en i onmen [ 29 , 32 ]. The p incipal pa hways ac i a ed by HIF- α a e angiogenesis, glycolysis, p og ammed cellula dea h, and pH egula ion o enhance he oxygen in he cells by inc easing he concen a ion o he oxygen anspo e (hemoglobin) by EPO o he oxygen low by ac i a ing he sympa he ic sys em [33,34] (Figu e 1). The le el o oxygen in bone issue is epo ed o be a ound 6.6–8.5% [ 35 ]. Bea ing his in mind, i is concei able ha exposu e o low concen a ions o oxygen can in luence bone cellula homeos asis h ough s imula ion o HIF [ 36 , 37 ]. The esul s desc ibed on he in luence o hypoxia on he main bone emodeling cells a e no uni o m and some imes con adic o y. This will depend on he me hodology used, as hey may a y depending on he deg ee o hypoxia o he hypoxia/ eoxygena ion models used. Taking in o accoun he me hodology used, gene ically, i may be ha hypoxia inhibi s he di e en ia ion and ac i a ion o he os eoblas s and induces he ac i a ion and ac i i y o os eoclas s. The e o e, he esul is an al e a ion o he bone mic oa chi ec u e, acili a ing a dec ease in s eng h and an inc eased isk o bone agili y ac u e. 3.1. Hypoxia and Os eoblas s I has been epo ed ha os eoblas ac i i y may dec ease in low oxygen en i on- men s. In his line, i has been shown ha os eoblas s cul u ed in a 2% oxygen en i onmen dec eased hei bone o ma ion ac i i y 10- old. The hypoxic en i onmen delayed he g ow h and di e en ia ion o os eoblas s [ 38 ], and os eoblas ogenesis was supp essed in sho - e m exposu e o a s o hypoxia [ 39 ]. Figu e 2shows he main e en s ha occu in os eoblas s in hypoxia. Hypoxia could be associa ed wi h educed Runx2 exp ession, causing a educ ion in he di e en ia ion o imma u e os eoblas s om mul ipo en mes- enchymal cell di e en ia ion [ 40 , 41 ]. The educed Runx2 could be caused by he exp ession o Twis ha is ac i a ed by HIF-1 α [ 42 ]. In addi ion, HIF-2 α impai s os eoblas di e - en ia ion by up egula ing Sox9, which inhibi s he exp ession o Runx2 and Sp7, ac o s in ol ed in os eoblas di e en ia ion [ 43 ]. Hypoxia has been associa ed wi h inhibi ion In . J. Mol. Sci. 2022,23, 3233 5 o 12 o he phospha idylinosi ol 3-kinase (PI3K)/Ak pa hway, which plays an impo an ole in an i-apop o ic and su i al signals in os eoblas s [ 44 ]. Hypoxia could also inhibi os- eoblas ogenesis, as i may inc ease induced SOST gene exp ession and he e o e inc ease he exp ession o scle os in in he os eocy es [ 45 ]. This is a con o e sial idea; i is also epo ed ha hypoxia dec eases scle os in exp ession [ 46 ]. Hypoxia is also associa ed wi h inhibi ion o os eoblas ma ix mine aliza ion. In he ma ix, mine aliza ion is c ucial in a se ies o pos - ansla ional modi ica ions o collagen ca ied ou by he PHD and lysyl oxidase oxygen-dependen enzymes, and hese enzymes a e educed in hypoxia [ 38 , 47 ]. I has been epo ed ha hypoxia educes he exp ession and ac i i y o alkaline phospha ase (ALP) [38]. Figu e 1. Response o hypoxia. The ac i i y o he p olyl hyd oxylase domain (PHD) and ac o - inhibi ing hypoxia (FIH) a e educed, a o ing hypoxia-inducible ac o (HIF)- α accumula ion and i s ansloca ion o he nucleus. In he cell nucleus, he HIF- α /HIF- β he e odime is o med. This o ms a complex ha binds o he hypoxia esponse elemen s (HRE) o HIF a ge genes and ac i a es he exp ession o mo e han 200 genes, ini ia ing he ac i a ion o cellula pa hways o enhance he oxygen in he cells. An agonis e ec o HIF-1 α on bone o ma ion is de e mined by he os eogenesis– angiogenesis coupling. Hypoxia s imula es inc eases in ascula endo helial g ow h ac o (VEGF), which p omo es os eo-angiogenesis and enhances bone o ma ion. Howe e , i is epo ed ha VEGF may induce an inc ease in he eso p i e ac i i y o os eoclas s [ 48 ]. Fu he mo e, os eoclas s a e s imula ed by adenosine iphospha e (ATP), which is eleased by os eoblas s du ing hypoxia [39]. In . J. Mol. Sci. 2022,23, 3233 6 o 12 Figu e 2. E ec s o hypoxia on os eoblas s. A hypoxic en i onmen is associa ed wi h a educ ion in bone o ma ion and os eoblas ma ix mine aliza ion. Hypoxia-inducible ac o (HIF)- α is associa ed wi h he exp ession o Twis , which down egula es Runx2 exp ession and causes a educ ion in os eoblas di e en ia ion. Runx2 exp ession is also educed by he exp ession o Sox9, which is p omo ed by HIF- α . Sox9 ac o also educes he exp ession o Sp7, ano he ac o in ol ed in os- eoblas di e en ia ion. Hypoxia has also been associa ed wi h inhibi ion o he phospha idylinosi ol 3-kinase (PI3K)/Ak pa hway, a o ing os eoblas apop osis. In a hypoxic en i onmen , in os eocy es, he e could be an inc ease in SOST gene exp ession and he e o e an inc ease in he exp ession o he scle os in p o ein, inhibi ing he Wn pa hway and hus, os eoblas di e en ia ion. In hypoxia, educed os eoblas ma ix mine aliza ion is associa ed wi h educed ac i i y o p olyl hyd oxylase domain (PHD), lysyl oxidase oxygen, and alkaline phospha ase (ALP). 3.2. Local E ec s o Hypoxia on Os eoclas s Os eoclas numbe s and ac i i y inc ease in a hypoxic en i onmen . I is epo ed ha os eoclas ac i i y is inc eased 21- old in exposu es o 2% o oxygen. In addi ion, i has also been epo ed ha 2% o oxygen inc eased eso p ion pi o ma ion 10- old [ 49 – 51 ]. Figu e 3shows he p incipal e en s p omo ed by hypoxia in he di e en ia ion, ma u a ion, and ac i i y o os eoclas s. I has been sugges ed ha PHD, HIF-1 α , and HIF-2 α play di ec oles in he eso p i e capaci y o os eoclas s. Hypoxia dec eases PHD, p omo ing he exp ession o p o- eso p i e genes ha i migh ini ially inhibi [ 52 ]. In addi ion, HIF-1 α ac i a es he exp ession o p o- eso p i e genes and glycoly ic ac i i y, s imula ing bone eso p ion [ 53 ]. Howe e , he eso p i e abili y o os eoclas s is inc eased because HIF-2 α inc eases mine al eso p ion by ac i a ing he exp ession o genes in ol ed in os eoclas ac i i y (TRAP, CTSK, and NFATC1) [54]. Hypoxia also p omo es os eoclas ogenesis: HIF-1 α and HIF-2 α ha e been associa ed wi h inc eased exp ession o os eoclas - usion- ela ed genes, accele a ing os eoclas cell usion [ 52 , 54 ]. Hypoxia has been associa ed wi h a supp ession o OPG, which a o s he RANK–RANKL in e ac ion and he NF- κ B pa hway [ 55 , 56 ]. TRAF6 is an adap e o RANK, p omo ing os eoclas ogenesis [ 57 ], and HIF-2 α may up egula e he exp ession o TRAF6 [ 54 ]. Os eoblas –os eoclas c oss alk plays an impo an ole in he esponse o os eoclas s du ing hypoxia. VEGF (syn hesized by os eoblas s du ing hypoxia) inc eases he eso p i e ac i i y o os eoclas s [ 48 ], which a e s imula ed by ATP, which is eleased by os eoblas s in a hypoxic en i onmen [39]. In . J. Mol. Sci. 2022,23, 3233 7 o 12 Figu e 3. E ec s o hypoxia on os eoclas s. Unde hypoxic condi ions os eoclas ogenesis is inc eased. Hypoxia-inducible ac o s 1 α and 2 α (HIF-1 α and HIF-2 α ) ac i a e os eoclas usion. HIF-2 α is also in ol ed in he exp ession o TRAF6, inc easing NF- κ B pa hway ac i a ion and os eoclas ma u a ion. NF- κ B is also ac i a ed because hypoxia dec eases os eop o ege in (OPG) exp ession. Hypoxia is associa ed wi h inc eased os eoclas eso p i e capaci y. P o- eso p i e gene exp ession is ac i a ed by HIF-1 α and HIF-2 α and by he inhibi ion o he p olyl hyd oxylase domain (PHD). HIF-1 α is also in ol ed in he s imula ion o glycoly ic ac i i y. In he os eoclas esponse o hypoxia, os eoblas – os eoclas c oss alk is c ucial. Reso p i e os eoclas ac i i y is s imula ed by ascula endo helial g ow h ac o (VEGF) and adenosine iphospha e (ATP); VEGF and ATP a e eleased by os eoblas s in a hypoxic en i onmen . 4. Hypoxia, E y h opoie in, and Bone Remodeling Du ing hypoxia, one o he genes a ge ed by he HIF- α /HIF- β complex is e y h opoi- e in (EPO), which encodes a ho mone c ucial o s imula ing ed blood cell p oduc ion. The EPO ecep o (EPO-R) is loca ed in e y h oid cells in he bone ma ow, and EPO binds wi h EPO-R o ac i a e he STAT3 and STAT5 pa hways o e y h opoiesis [58]. The e a e se e al epo s ha associa e EPO- ela ed diseases and al e a ions in bone me abolism [ 59 , 60 ]: in his scena io i has been epo ed ha EPO s imula es os eogenesis. Os eogenesis due o EPO could be associa ed wi h he exp ession o EPO-R in os eoblas s and he ac i a ion o he mammalian a ge o he apamycin (mTOR), JAK2, and PI3K pa hways [ 61 , 62 ]. The exp ession o EPO is associa ed wi h angiogenesis ac i a ion which, as desc ibed abo e, is c ucial in os eoblas –os eoclas c oss alk du ing hypoxia [ 63 , 64 ]. In addi ion, i is also epo ed ha EPO could ac i a e he di e en ia ion o os eoblas s h ough di ec in e ac ion wi h os eoblas p ecu so s o by s imula ing BMP p oduc ion [ 61 , 62 , 65 ]. Howe e , EPO could be associa ed wi h educed os eoblas mine aliza ion [ 66 , 67 ] h ough he bone–kidney–pa a hy oid gland axis [ 68 ]. Pa adoxically, an associa ion be ween EPO and inc eased bone eso p ion has been epo ed. Os eoclas ogenic ac i i y is also ac i a ed by he JAK2 and PI3K signaling pa hways [ 67 , 69 ]. EPO has been associa ed wi h he di e en ia ion o p e-os eoclas s o ma u e os eoclas s [ 70 ]. The disc epancies be ween bone o ma ion and bone eso p ion associa ed wi h EPO a e poo ly unde s ood bu may esul om la ge di e ences in he expe imen al models used. Ne e heless, i seems clea ha EPO plays a ole in modula ing bone cell esponses unde hypoxic condi ions. 5. Vi amin D Me abolism, In lamma ion, Hypoxia, and Bone Vi amin D is c ucial in phospho ous and calcium me abolism, cell p oli e a ion, and he con ol o inna e and adap a i e immuni y. The i amin D ecep o (VDR) is ac i a ed In . J. Mol. Sci. 2022,23, 3233 8 o 12 by he binding o calci iol (1-al a, 25-dihid oxicolecalci e ol) o o m a complex wi h he e inoid X ecep o (RXR). The calci iol/VDR/RXR complex mig a es o he nucleus o ac i a e he exp ession o genes in ol ed in i amin D signaling [ 71 ]. In his scena io, i is sugges ed ha hypoxia-associa ed al e a ions in bone me abolism may also be media ed by i amin D/VDR signaling. In immune cells, i is known ha HIF-1 α ac i a es he exp ession o cy okines such as in e leukin-1 β (IL-1 β ), IL-6, umo nec osis ac o - α (TNF- α ), and in e e on-gamma (IFN- γ ), p omo ing an in lamma o y esponse o hypoxia [ 72 , 73 ]. Ac i a ion o he ansc ip ion o in lamma o y- ela ed genes is h ough NF- κ B pa hway ac i a ion by HIF-1 α [ 74 ]. In os eoclas s, he NF- κ B pa hway can also be ac i a ed by HIF- α [ 54 ]. In non-s imula ed cells, he NF- κ B pa hway is no ac i a ed. The p65 componen o NF-kB in he cell cy oplasm binds o IkB p o eins. A e cell s imula ion, he IkB p o eins a e phospho yla ed and ubiqui ina ed o be deg aded ia he p o easome pa hway. This allows he p65 componen o NF- κ B o be ansloca ed o he nucleus, ac i a ing he exp ession o a ge genes o he NF- κ B pa hway [ 75 ]. In his scena io, i is epo ed ha calci iol inhibi s p65 ansloca- ion o he nucleus and he phospho yla ion o he IkB p o ein [ 76 ]. Thus, i amin D has been associa ed wi h a educ ion in NF- κ B pa hway ac i a ion in immune cells and os eo- clas s [ 77 – 79 ]. In addi ion, in os eoclas s, i amin D can supp ess HIF-1 α exp ession [ 80 ] and TNF-αdown egula es he exp ession o VDR [79]. 6. Hypoxia and Bone Remodeling in Clinical S udies In humans, he e is no good model o he e ec s o ch onic hypoxia on bone me abolism and, in addi ion, he e a e o he associa ed ac o s. In his scena io, con adic o y e- sul s ha e been epo ed. An associa ion be ween long- e m sus ained hypoxia exposu e and a educ ion in se e al indices o bone heal h has been epo ed [ 7 – 9 ]. Fo example, Basu e al. (2013) s udied membe s o he Indian a my o ou mon hs a al i udes o 5400–6700 m . In con as , wi h sho exposu es, whe e no al e a ions we e obse ed [ 81 ], his epo s udied humans a a simula ed 4000 m al i ude o 21 days. As he e is no good model analyzing he e ec s o ch onic hypoxia in clinical s udies, one op ion could be obs uc i e sleep apnea synd ome, in which noc u nal hypoxia plays a key ole. Howe e , i mus be aken in o accoun ha in e mi en hypoxia has di e en pa hophysiological mechanisms. Bone emodeling ma ke s ha e a diu nal/noc u nal a ia ion. Reso p ion ma ke s inc ease du ing he nigh , wi h a peak in he ea ly mo ning, and dec ease in he la e a e noon. In he case o he o ma ion ma ke , i is simila bu less in ense [ 82 ]. Tomiyama e al. (2008) desc ibed a link be ween obs uc i e sleep apnea and inc eased bone eso p ion, and ound a posi i e ela ionship be ween apnea/hypopnea episodes and u ina y ca boxy- e minal collagen c osslinks (CTX) [ 83 ]. A me a-analysis o 112,258 subjec s epo ed ha sleep apnea synd ome was a isk ac o o os eopo osis [ 84 ]. Indi idual s udies showed an associa ion be ween sleep apnea synd ome and he p ese a ion o bone mine al densi y (BMD) [ 85 , 86 ]. Obs uc i e sleep apnea has also been associa ed wi h he isk o agili y ac u e [ 87 ]. Analyzing he hypoxic consequences in human s udies, i seems clea ha hypoxic condi ions can modi y bone emodeling, wi h he le el o expo- su e, ime, and equency being c ucial. Fu u e esea ch should ocus on unde s anding he molecula esponse o hypoxia and i s consequences in bone emodeling. 7. Conclusions The molecula esponse o hypoxia is media ed by HIF. In bone, hypoxia has been associa ed wi h dec eased os eoblas di e en ia ion and ac i i y and inc eased os eoclas ma u a ion and ac i i y. Hypoxia is associa ed wi h educed bone o ma ion and educed os eoblas ma ix mine aliza ion due o he hypoxia en i onmen inhibi ing os eoblas di e en ia ion (mainly media ed by Runx2, Sox9, Wn , and PI3K/Ak signaling pa hways). A hypoxic en i onmen is associa ed wi h inc eased os eoclas ogenesis and os eoclas bone eso p i e capaci y. EPO and i amin D me abolism could play a key ole in modula ing he bone molecula esponse o hypoxia. In he case o clinical s udies, con adic o y esul s In . J. Mol. Sci. 2022,23, 3233 9 o 12 ha e been published, al hough i seems clea ha a hypoxic en i onmen can modi y bone me abolism. In his scena io, u he esea ch is necessa y o unde s and he bone molecula esponse o hypoxia and i s possible clinical consequences on bone me abolism. Au ho Con ibu ions: Concep ualiza ion: R.U.-M. and J.L.P.-C.; Me hodology: R.U.-M., R.R., M.R.-M. , J.-M.F.-G., A.D. and J.L.P.-C.; w i ing—o iginal d a p epa a ion: R.U.-M. and J.L.P.-C., w i ing— e iew and edi ing: R.U.-M., R.R., M.R.-M., J.-M.F.-G., A.D. and J.L.P.-C. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This esea ch ecei ed no ex e nal unding. Ins i u ional Re iew Boa d S a emen : No applicable. 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