Epigenetic Regulation of Autophagy in Bone Metabolism

Abstract The skeletal system is crucial for supporting bodily functions, protecting vital organs, facilitating hematopoiesis, and storing essential minerals. Skeletal homeostasis, which includes aspects such as bone density, structural integrity, and regenerative processes, is essential for normal skeletal function. Autophagy, an intricate intracellular mechanism for degrading and recycling cellular components, plays a multifaceted role in bone metabolism. It involves sequestering cellular waste, damaged proteins, and organelles within autophagosomes, which are then degraded and recycled. Autophagy’s impact on bone health varies depending on factors such as regulation, cell type, environmental cues, and physiological context. Despite being traditionally considered a cytoplasmic process, autophagy is subject to transcriptional and epigenetic regulation within the nucleus. However, the precise influence of epigenetic regulation, including DNA methylation, histone modifications, and non-coding RNA expression, on cellular fate remains incompletely understood. The interplay between autophagy and epigenetic modifications adds complexity to bone cell regulation. This article provides an in-depth exploration of the intricate interplay between these two regulatory paradigms, with a focus on the epigenetic control of autophagy in bone metabolism. Such an understanding enhances our knowledge of bone metabolism-related disorders and offers insights for the development of targeted therapeutic strategies.


Introduction
2][3] Maintaining bone homeostasis is paramount for ensuring the normal functionality of the skeleton, encompassing critical aspects such as bone density, structur al inte grity, and re gener ative processes. 4utophagy sequesters cellular waste, damaged proteins, and organelles within autophagosomes, subsequently subject to de gr adation and recycling. 5The outcome of autophagy in bone meta bolism de pends on v arious factors, including autopha gy re gulation, cell type , environmental cues, and physiological context. 6][10] However, the precise impact of epigenetic regulation, encompassing DNA methylation, histone modifications, and non-coding RN A (ncRN A) expression, on cellular fate r emains incompletel y elucidated. 8The interplay between autophagy and epigenetic modifications adds a layer of complexity to bone cell regulation.
This article explores the intricate interplay between these tw o re gulatory par adigms, shedding light on the epigenetic contr ol of autopha gy within the r ealm of bone metabolism.This exploration enhances our comprehension of bone metabolismrelated disorders and pondered the future and challenges of epigenetic regulation of autophagy in bone metabolism.

Overview of Epigenetic Modifications
Epigenetics br oadl y pertains to inherita b le modifications in phenotype that do not inv olv e modifications in DNA sequences. 11 , 12hile the genotype remains unchanged, her editar y alterations occur in the phenotype.4][15] These mechanisms serve as key regulators of gene expression, functioning either through the modulation of chromatin structure and gene transcription or, in the case of ncRNAs, through posttranscriptional control of protein translation. 16 , 17Epigenetics plays a r egulator y r ole in various biological processes, including the specific expression of genes in different tissues, the inacti v ation of chr omosomes, genomic imprinting, and the differentiation of cells. 18 , 19With the advancement of resear c h techniques, e pigenetic a bnormalities hav e been implicated in causing malignancies, metabolic disorders, somatic diseases, and autoimmune diseases. 20Epigenetics highlights the intricate interplay between genetic and environmental factors and plays a crucial role in regulating bone metabolism, 21 particularly in the differentiation of bone cells, providing insights into the study of bone metabolic disorders and their treatment directions.

DN A Methyla tion
DNA methylation stands as one of the most extensi v el y studied and common epigenetic modifications in mammals. 22This entails methylating the 5 carbon of cytosine, leading to the formation of 5-methylcytosine (5-mC). 23 , 24Cytosine methylation pr edominantl y occurs within specific dinucleotide sequences known as CpG sites.Importantly, these CpG sites are not randoml y distributed acr oss the genome but ar e concentrated in regions called CpG islands, often situated at the 5 ends of genes.High meth ylation le v els in CpG islands ar e commonl y associated with gene silencing, while low methylation levels are linked to gene acti v ation. 25 , 26he enzymes r esponsib le for transferring methyl gr oups from S -adenosylmethionine (SAM) to DN A include DN A methyltr ansfer ase 1 (DNMT1), DNMT3a, DNMT3b, and DNMT3L. 27emethylation of DNA can occur either passi v el y following DNA r e plication, leading to reduced maintenance methylation, or acti v el y. 28 The pr ecise molecular mechanisms underl ying acti v e demethylation are not yet fully understood.For instance, in the context of bone biology, Interferon Regulatory Factor 8 (IRF8) transcription factor inhibits osteoclast formation. 29Experimental evidence has r ev ealed that DNMT3a plays a significant role in inhibiting the activity of IRF8 by increasing methylation at distal r egulator y elements associated with the IRF8 gene.Elevated levels of SAM can enhance this methylation, ultimately promoting osteoclast differentiation and bone resorption. 30Deleting DNMT3a in osteoclasts (OC) or using the inhibitor TF-3 in mice pr otects a gainst bone loss after ov arian r emov al. 31 Mor eov er, in multiple m y eloma patients, alter ations in bone resorption have been associated with elevated IRF8 methylation, induced by m y eloma cells' r elease of thymidine phosphor ylase (TP), leading to decreased IRF8 expression and enhanced bone resorption. 32he transcription factors runt-related transcription factor 2 (Runx2) and Osterix (OSX) play critical roles in regulating osteob last (OB) differ entiation and bone matrix synthesis.Runx2 acti v ates the pr omoter of OSX, indicating Runx2's position as an upstream transcription factor in osteogenesis. 33During mesenchymal stem cell differentiation into osteoblasts, there is a reduction in the methylation level of Runx2, underscoring the significant r egulator y function of Runx2 methylation during osteob last differ entiation. 29Additionall y, e pigenetic r egulation of OSX is vital for guiding mesenchymal stem cells (MSCs) into osteob last differ entiation. 34he Ten-Elev en-Translocation (TET) famil y of enzymes ar e recognized for their ability to remove methyl groups from DNA, converting 5-mC into 5-hydroxymethylcytosine (5-hmC).Demeth ylation mediated b y TET enzymes enhances chromatin accessibility for target genes controlled by Runx2, facilitating tr anscriptional re gulation.TET proteins engage with Runx2 via their catalytic domain, influencing cytosine methylation patterns around the Runx2 binding region. 35Disruptions in the promoter methylation of key bone-related genes, such as bone morphogenetic protein 2 (BMP2), may lead to irregularities in bone formation. 29

Post-Tr ansla tional Histone Modifications
Inside the eukaryotic nucleus, a segment of double-stranded DNA spanning 147 base pairs coils around eight histone proteins, which include two copies of H2A, H2B, H3, and H4, cr eating n ucleosomes.7][38] The N-terminal amino acid tails of H3 and H4 undergo post-translational modifications that pr ofoundl y affect both chromatin structure and DNA-associated activities. 39Resear c h has unveiled that euc hr omatin, r e pr esenting a relaxed and actively tr anscribed DN A state , is marked by elev ated lev els of acetylation and trimethylation at H3K4, H3K36, and H3K79.In contrast, reduced acetylation levels and heightened methylation at H3K9, H3K27, and H4K20 indicate a more condensed, transcriptionally inactive heterochromatin structure. 23 , 40Histone modification is the most intricate mode of regulation among the three epigenetic modifications. 41Curr entl y, the most extensi v el y studied histone modifications in bone metabolism are histone acetylation and histone methylation.

Histone Acetylation
Histone acetylation inv olv es the addition of acetyl groups to histones and is governed by histone acetyltr ansfer ases (HATs).Conv ersel y, histone deacetylases (HDACs) regulate the removal of these acetyl groups, leading to histone deacetylation. 42This balance between HATs and HDACs maintains a delicate equilibrium.
Histone acetyltr ansfer ases facilitate the attachment of acetyl-CoA molecules onto histones, promoting a relaxed nucleosome structure. 43This structural change acti v ates the transcriptional machinery, enhancing gene expression.In cases wher e HAT acti vity is hinder ed or inhibited, the r e pair of damaged DNA may be compromised, potentially resulting in cellular apoptosis or programmed cell death.This underscores the crucial role of HATs in DNA repair and cell survival. 44Unlike HATs, HDACs r emov e acetyl gr oups fr om histones, causing histones to tightly bind to negatively charged DNA.This leads to dense chromatin compaction and the inhibition of gene transcription. 45istone acetyltr ansfer ases are classified into distinct subfamilies based on their catalytic domains structural and functional similarities, r e pr esented as HAT domains.These subfamilies include p300/CBP, HAT1, MYST, PCAF/Gcn5, and Rtt109. 46onv ersel y, HDACs are categorized into 4 classical classes according to their sequence similarities: Class I, Class II, Class III, and Class IV. 47 The Class I, II, and IV HDACs contain a conventional deacetylase domain, while Class III HDACs feature a NAD + -de pendent catal ytic domain ( Ta b le 1 ). 48he transcriptional activity of the Runx2 gene is modulated by the acetyltr ansfer ase P300 and nicotinamide phosphoribosyltr ansfer ase (Nampt).P300 enhances osteogenic differentiation in MSCs through H3K14 acetylation, while Nampt does so in MC3T3-E1 cells via H3K9 acetylation. 49 , 50Other acetyltr ansfer ases, GCN5 and PCAF, which acetylate histone H3K9, are known to enhance osteogenic differentiation and bone formation by acetylating H3K9 loci in the promoters of Wnt and BMP genes.However, their levels have been found to decline significantly in mice with ovariectomy-induced osteoporosis (OVX). 51 , 52In contrast to acetyltransferases, 2 HDACs, HDAC1 and Sirtuin1 (SIRT1), play roles in osteogenesis.HDAC1 promotes MSC osteogenesis through the deacetylation of Forkhead box O3a (FoxO3a), while SIRT1 inhibits osteogenic differentiation in Bone Marrow Stromal Cells (BMSCs) by deacetylating JAGGED1 (JAG1). 53 , 54n terms of bone health, n umer ous studies indicate that HDA Cs, particularly SIR T, play a significant role in bone development.They influence processes such as bone formation, r e pair, and re gener ation. 55SIRT1, a r e pr esentati v e of Class III HDACs, acti v ation induces autopha gy during cellular str ess, dir ectl y deacetylating autopha gy-r elated pr oteins (A TG5, A TG7, LC-3) to initiate autophagy. 56 , 57SIRT1 also deacetylates FoxO3, a transcription factor for autopha gy-r elated genes. 58SIRT1 plays a pivotal role in driving MSC differentiation to war d osteoblasts. 59 , 60stone Methylation Histone methylation mainly happens at lysine (K) and arginine (R) residues found at histones' N-terminal regions.In contrast to acetylation, meth ylation e vents at these sites contribute to both transcriptional acti v ation and inhibition.For instance, trimethylation of H3K4, H3K36, and H3K79 is associated with  gene acti v ation, while trimethylation of H3K27, H3K9, and H4K20 is linked to gene r e pr ession.71 , 72 Histone methylation is dynamically regulated by methyltr ansfer ases and demethylases.73 , 74 Methylation enzymes such as Suppressor of Variegation 3-9 Homolog 1 (SUV39H1), G9a, and Enhancer of Zeste Homolog 2 (EZH2) add methyl groups, while demethylation enzymes such as Lysine-specific demethylase 1 (LSD1) and JmjC domain (JMJD)-containing pr oteins r emov e methyl groups.
Methyltr ansfer ases and demethylases play pi v otal r oles in regulating gene expression in osteoblasts and osteoclasts, thereby influencing the functioning of related genes.( Ta b le 2 ) For instance, SUV39H1/2 methyltr ansfer ases pr edominantl y regulate the abundance of trimethylated H3K9.Knockdown of SUV39H1 leads to a reduction in H3K9me3 levels, enhancing DNA r e pair capa bilities and delaying cellular senescence in pr oger oid cells. 75xcessi v e EZH2 activity leads to elevated H3K27me3 levels, causing a shift in the lineage commitment of BMMSCs to war d adipocytes during osteoporosis. 76Further investigations reveal significant enrichment of both EZH2 and H3K27me3 in the promoters of Wnt1, Wnt6, and Wnt10a within BMMSCs of mice subjected to ovariectomy. 77 , 78Notably, EZH2 reduces the enrichment of H3K27me3 on these pr omoters, consequentl y suppr essing the expression of Wnt genes.Overexpressing EZH2 results in heightened H3K27me3 levels at the transcription start sites (TSS) of Runx2 and Bglap, pi v otal factors initiating osteogenesis.
The histone methyltr ansfer ase SET-domain-containing 2 (SETD2) catalyzes the modification of H3K36 trimethylation.It facilitates the binding of trimethylated histones to promoters associated with lipopol ysaccharide-binding pr otein (LBP), thus influencing the specification of adipogenic and osteogenic pathways.Disrupting SETD2 through knockout shifts the fate of mesenchymal stem cells to war d adipoc yte formation, impairing their potential to differentiate into osteoblasts.In mice lacking Setd2 specifically in osteoprogenitor cells, there is a notable decr ease in tra becular v olume and bone formation r ate , accompanied by an excessi v e accum ulation of marr ow fat. 79MJD3 functions as a demethylase targeting H3K27.JMJD3 plays a r egulator y r ole in influencing the expr ession of genes related to bone health, such as nuclear factor of acti v ated T-cells cytoplasmic 1 (NFATC1), 80 Runx2, OSX, osteopontin, bone sialoprotein (BSP), and osteocalcin (OCN). 79 , 81In vitro experiments r ev eal that inhibiting LSD1 through knockdown using shRNA or pharmacological inhibitors suppresses osteoblast function and differentiation.When LSD1 activity is inhibited in vivo, it leads to a reduction in both osteoblast count and activity, consequentl y r esulting in osteopenia.Selecti v e elimination of LSD1 from mesenchymal cells also results in osteopenia and disturbs the structure of the growth plate.

Non-Coding RNAs
Non-coding RNAs constitute a significant portion of the human genome, with appr oximatel y 2% consisting of pr otein-coding genes.The majority of the genome is made up of non-proteincoding RNAs, 91 , 92 involving long non-coding RN As (lncRN As), cir cular RN As (cir cRN As), and small non-coding RNAs (sncR-NAs).Within the category of sncRNAs, there are further subdivisions, such as microRNAs (miRNAs), piwi-interacting RNAs (piR-NAs), small-interfering RNAs (siRNAs), and more.These ncRNAs play crucial roles in gene expr ession, cell differ entiation, dev elopment, and various diseases, making them a prominent area of study in modern biology. 93

Long Non-Coding RNAs
Long Non-Coding RNAs (LncRNAs) are ncRNAs that consist of more than 200 nucleotides in length.They have emerged as noncanonical regulators participating directly in various pathophysiological processes, including autophagy. 94s resear c h ad vances, increasing evidence suggests that lncRNAs, as key regulators of gene expression, play pivotal roles in the proliferation, differentiation, apoptosis, and activity of osteoblasts and osteoclasts ( Table 3 ).Disruptions in their expr ession patterns hav e been linked to n umer ous diseases, such as aging, cancer, metabolic disorders, and osteoporosis (OP).
Long non-coding RNAs also play dual roles in osteoclastogenesis.For example, MIRG has been shown to have a positi v e r egulator y r ole, 109 while NEAT1 110 , Bmncr, 110 NRON, 111 and others inhibit osteoclast formation by various mechanisms.Overall, lncRNAs r e pr esent a di v erse class of r egulator y molecules with complex roles in the regulation of osteoblasts and osteoclasts, influencing bone health and associated diseases.

MicroRNAs
MicroRN As (miRN As) are short, ncRN A molecules typically composed of 19 to 25 nucleotides in length.They are highly conserv ed acr oss species and play a crucial r ole in posttr anscriptional gene re gulation. 120MicroRN As do not code for pr oteins themselv es but instead regulate gene expression by binding to complementary sequences within the target mRNA, leading to the de gr adation of the target mRNA or translational r e pr ession. 121The intricate interplay between miRNAs and their target mRNAs is influenced by various factors, including the strength of their interaction, target mRNA abundance, and intracellular localization of both miRNA and mRNA. 122In the context of osteogenesis and bone cell differentiation, miRNAs ha ve se veral ke y r oles: r egulation of bone formation, di v erse functions and mechanisms, targeting multiple genes and potential therapeutic targets ( Ta b le 4 ).
Over all, miRN As are essential regulators of osteogenesis and bone cell differentiation, contributing to the maintenance of bone tissue and the equilibrium between bone formation and resorption.Understanding the roles and regulatory networks of miRNAs in bone biology is crucial for advancing our knowledge of bone-related diseases and potential therapeutic interventions.

Autophagy in Bone Metabolism
In mammals, three specific types of autopha gy hav e been r ecognized: macr oautopha gy, micr oautopha gy, and chaperonemediated autophagy.Of these, macroautophagy is the most pr ev alent and is intricately intertwined with cellular physiology, biological functions, and the development of diseases within the context of bone. 134In this re vie w, we will primarily focus on macr oautopha gy when referring to autophagy.
Autopha gy typicall y pr ogr esses thr ough four key sta ges: the creation of autophagosome precursors, followed by the formation of autophagosomes, then the development of autolysosomes, and finally, the de gr adation phase .During the de gr adation phase, siza b le cellular molecules ar e disinte gr ated into amino acids, lipids, nucleotides, and energy, facilitating both the cell's metabolic necessities and the rejuvenation of specific organelles. 135urr entl y, ther e ar e mor e than 40 ATG genes known to r egulate autopha gy. 136Each gene plays distinct roles at specific stages.ULK1, a key protein, forms the ULK1 complex (ULK1-A TG13-FIP200-A TG101) initiating autophagy.During starvation, mTOR inhibition and AMPK acti v ation pr ompt ULK1 phosphor ylation, initiating autopha gy.Beclin1, similar to yeast ATG6, interacts with proteins to form the PI3K complex, crucial for autophagy initiation.ATG14 and UVRAG play roles in autophagosome elongation and autopha gosome-l ysosome fusion, r especti v el y.Pr oteins such as ATG12, ATG5, and LC-3 control autophagosome formation.LC3-II, a key autophagy marker, encapsulates materials for de gr adation.F inally, the autophagosome fuses with lysosomes to form autolysosomes, where de gr adation pro vides cells with ener gy a gainst str ess. 137

Importance of Autophagy in Bone Homeostasis and Remodeling
Bone homeostasis refers to the delicate balance maintained within the skeletal system through a dynamic interplay between bone-forming cells (osteob lasts), bone-r esorbing cells (osteoclasts), and bone matrix. 138This intricate equilibrium is crucial for preserving the overall health, strength, and functionality of bones ( Figure 1 ).Bone r emodeling inv olv es thr ee cell types: MSCs differentiate into osteoblasts (OB) on the bone surface, secreting bone matrix.This matrix, with OB, transforms into osteocytes, forming a vital mec hanosensory netw ork in bones, essential for signaling; sim ultaneousl y, m ultin ucleated osteoclasts (OC) deri v ed fr om hematopoietic stem cells constantly break down and absorb the neighboring bone matrix. 138 , 139Generally, the balance between bone formation and resorption is continually coordinated.In this manner, the quality, structure, and function of bone tissue can be influenced by internal or external stimuli.Autophagy helps OB, OC, and chondrocytes cope with stress and nutrient deficiencies, promoting survival in harsh hypoxic and hypertonic conditions.Autophagy can also enable the long-term terminal differentiation of osteocytes.The process of autophagy encompasses not only the osteoclastic resorption process but also the acquisition of energy sources during osteoblast differentiation. 140Alter ed autopha gy can disrupt bone cell balance, potentially causing various diseases.

Autopha g y in BMSCs
As a rare and di v erse subset of stromal cells, BMSCs exhibit the capacity for both self-r enew al and differ entiation.These cells ar e capa b le of undergoing differ entiation into v arious linea ges, not limited solely to mesenchymal lineages such as osteocytes, chondroc ytes, and adipoc ytes, 141 , 142 autophagy is vital in contr olling the r oles, differ entiation, and survi v al of BMSCs.Modulating autophagic activity in these stem cells could have implications for enhancing bone re gener ation, treating bone-related diseases, and addressing age-related bone health issues.
Autopha gy-r egulated r edox state participates in determining the fate of BMSC differentiation.It has been r e ported that elev ated R OS lev els in BMSCs can promote adipogenesis while inhibiting osteogenic differentiation.When excessi v e R OS is generated within cells, the autophagic mechanism is acti v ated to reduce ROS le vels, thereb y restoring osteogenic differentiation of BMSCs. 143Additionall y, r esear c h has demonstr ated that administering the neur ope ptide substance P (SP) to rats enhances BMSC autophagic activity through the AMPK and mammalian target of r apam ycin (mTOR) pathways, concurr entl y r educing R OS pr oduction and facilitating osteogenic differentiation. 144Rapamycin (RAPA), a well-known inhibitor of the mTOR, triggers autophagy by binding to mTOR and acti v ating the mTOR signaling pathway.Resear c h has indicated that RAPA enhances autophagy and influences the osteogenic differentiation of MSCs. 145

Autopha g y in Osteoblast
Osteob lasts ar e central to the processes of bone gr owth, r e pair, and remodeling.They synthesize collagen proteins and other bone matrix molecules, providing structural support to the skeleton.Osteoblasts also release bone formation-related proteins, hormones, and cytokines, such as alkaline phosphatase, osteocalcin, and others, which play significant roles in regulating bone metabolism and maintaining bone homeostasis. 146utopha gic pr oteins such as Beclin1, A TG5, and A TG7 play an essential role in facilitating the mineralization of osteoblast cell lines.Osteob last autopha gy deficiency can reduce its mineralization ability, leading to a low bone mass phenotype. 147nsufficient or deficient autophagy in osteoblasts leads to an increase in oxidative stress, which in turn elevates the production of TNFSF11.This further enhances the differentiation of osteoclasts, ultimately resulting in a phenotype resembling osteoporosis. 148Transcription factors FoxO and ATF4 have been extensi v el y studied in the regulation of autophagy during the differentiation and function of osteoblasts.FoxO binds to the pr omoter r egions of autopha gy genes, incr easing autopha gic activity.This activation may promote the differentiation of MSCs into osteoblasts and inhibit fat generation. 149Additionally, under endoplasmic reticulum (ER) stress and amino acid deficiency conditions, ATF4 can pr omote autopha gosome formation and autophagic flux by regulating the expression of autopha gy initiation-r elated genes, contributing to the maintenance of osteoblast homeostasis. 150

Autopha g y in Osteoclasts
Osteoclasts primarily engage in bone resorption in the body and play a role in bone homeostasis.When their activity is excessi v el y high, it can lead to osteoporosis, while conversely, decr eased acti vity can r esult in incr eased bone formation. 151he micr oenvir onment wher e osteoclasts ar e distributed, such as the sealing zone and the interior of bone tr abeculae , is c haracterized by low oxygen levels, which support the survi v al and maturation of osteoclasts. 152Under low oxygen conditions, the expression of Hypoxia-inducible factor 1 alpha (HIF-1 α) and its downstream signaling molecule BNIP3 increases, leading to elev ated lev els of autopha gy-r elated pr oteins such as A TG5, A TG12, and Beclin1.Consequently, LC-3 is recruited to autophagosomes, enhancing the expression of bone resorption factors such as RANKL, matrix metalloproteinase K (MMP), tissue protease K, NFATc1.This, in turn, leads to increased osteoclast formation. 153 , 154Resear c h has provided evidence that the targeted r emov al of ATG7 in osteoclast precursors in mice resulted in the impr ov ement of bone loss and the excessi v e acti v ation of osteoclasts triggered by glucocorticoids or ovariectomy. 155uring the adhesion and migration of osteoclast precursor cells, chemotactic factors CXCL12 and S1P play roles in this process.Specifically, S1P binds to its receptor S1PR on the membrane of osteoclast precursor cells, participating in the migration process. 156S1P also modulates autophagy through mTOR, serving as a link between autophagy and the accumulation of osteoclasts. 157RANKL and RANK (r ece ptor acti v ator of NF-κB) play essential roles in osteoclast differentiation and maturation.RANKL induces autophagy activation through pathways such as MAPK and NF-κB during this process. 158

Autopha g y in Osteocytes
Osteocytes esta b lish an extensi v e inter connected netw ork throughout the entire skeleton.They do this through multiple br anc hing processes that resemble dendrites, allowing them to connect with other types of bone cells such as osteoblasts, bone lining cells, and stromal cells. 159This network spans from the innermost bone regions to the blood vessel linings. 160Osteocytes play m ultifaceted r oles: they r e gulate miner al metabolism and the remodeling of the perilacunar matrix, while also serving as mechanosensory cells. 161steocytes play a central role in regulating bone remodeling in response to mechanical loading. 162Autophagy is a vital mechanism that ensures the survi v al of osteocytes and enhances their capacity for mec hanotr ansduction in the context of bone remodeling.Recent in vestigations ha ve un veiled autophagy's r esponsi v eness to mechanical cues in osteocytes.Mechanistically induced autophagy contributes to the preservation of adenosine triphosphate (ATP) and fosters osteocyte survi v al. Furthermor e, gi v en the unique featur es of terminal differ entiation, the prolonged lifespan of bone cells, and their oxygen and n utrient-de pri v ed envir onment, autopha gy becomes a key player in the regular physiological processes of these cells. 140 , 163nsurprisingly, osteocytes exhibit a notable baseline level of autophagy both in laboratory settings and within living organisms.Selecti v el y deleting the autopha gy-r elated gene ATG7 in osteocytes inhibits autophagy, causing decreased bone formation and reduced bone mass in young adult mice, r esemb ling the effects of aging on the skeletal system. 164ysr egulated autopha gy in osteocytes, as seen in Ephrin B2 deficiency or triggered by substances such as pinocembrin, can affect bone health and apoptotic processes. 165 , 166Beyond its role as a de gr adati v e mechanism, recent evidence has spotlighted the inv olv ement of autopha gy in pr otein secr etion, r eferr ed to as secr etor y autopha gy, bridging intracellular autophagy with the extracellular micr oenvir onment. 167This phenomenon may offer insights into understanding the impacts of osteocyte autopha gy trigger ed b y ph ysical forces exerted on osteoblasts and osteoclasts. 168

Autopha g y in Chondrocytes
MSCs in the bone marrow differentiate into osteoprogenitor cells, which further differentiate into chondrocytes that form the cartilage primordia. 169 , 170Con versely, the y de velop into endoc hondr al bone , determining the r ate and length of longitudinal bone growth.Most skeletal growth is achieved through the ossification of cartilage in the epiphyseal growth plate of bones.Due to the low re gener ative capacity of chondrocytes and limited vascularity within the growth plate, chondrocytes ar e pr one to hypoxia and n utrient deficiency. 171It has been demonstrated that inflammatory mediators including reactive oxygen species (ROS), IL-1 β, nitric oxide (NO), Fas, and tumor necrosis factor-alpha (TNF-α) are strongly associated with chondrocyte apoptosis. 172utophagy, a cellular de gr adation mec hanism r esponsib le for maintaining cellular energy metabolism homeostasis, possesses the ability to restore impaired chondrocyte functionality. 173When cellular ATP levels decrease, AMPK activates and triggers autophagy, restoring nutrients and ATP, thus maintaining cellular energy balance.Autophagy also impacts protein and lipid metabolism in chondrocytes through the mTOR pathway. 174t has been demonstrated that chondrocyte degeneration and apoptosis are considered primary factors in the development of osteoarthritis (OA). 175Consequently, numerous experiments have been designed to enhance chondrocyte autophagy as a means to alleviate OA.For instance, Interventions such as vitamin D and tofacitinib exhibit potential in pr otecting a gainst OA by promoting autophagy and preventing chondrocyte degeneration. 176 , 177Proteins such as PGRN and G protein-coupled r ece ptor famil y C gr oup 5 member B (GPRC5B) play roles in maintaining chondrocyte health through their involvement in autophagy. 178 , 179Moder ate mec hanical str ain can promote the

Epigenetic Regulation of Autophagy in Bone Metabolism
Epigenetic modifications, alterations in gene expression without DN A sequence c hanges, 11 intersect significantly with autopha gy, a pr ocess vital for cellular str ess r esponse and r ecycling.This intricate r elationship pr ofoundl y influences bone metabolism and homeostasis.

Epigenetic Modifications and Autophagy
DNA methylation, a herita b le e pigenetic modification, impacts autopha gy r egulation in di v erse cancers and stem cells. 173ypermethylation of LC-3A 174 and Beclin1 175 inhibits autophagy, pr omoting tumorigenesis.Conv ersel y, demethylation enhances autophagy, showing therapeutic potential in cancer treatments. 176 , 178 , 179Additionally, in conditions like osteoporosis and osteoarthritis, abnormal DNA methylation affects autophagy, highlighting its role in bone health. 180istone modifications such as methylation and acetylation regulate autophagy-related genes.G9a-mediated histone methylation inhibits autophagy, while its dissociation activates this process, crucial in non-alcoholic fatty liver disease. 181nzymes such as JMJD3 182 , 183 and SIRT 184 , 185 family members modulate histone demethylation and deacetylation, respecti v el y, impacting autophagy regulation in cellular processes.

Epigenetic Regulation of Autophagy in Bone Metabolism-Related Diseases
Epigenetic mechanisms are crucial in regulating autophagy processes, influencing its transcriptional and post-translational regulation. 195 , 196These epigenetic mechanisms can be further influenced by external stimuli, alterations in phenotypic states, or pathological environmental conditions. 197Epigenetic modifications and autophagy interact in bone metabolism, regulating gene expression, cellular clear ance , and stress responses, crucial for maintaining bone tissue homeostasis and function.The convergence of genetic and environmental factors, causing disruption in the e pigenetic r egulation of autophagy, has the potential to detrimentally affect bone metabolism, thereby contributing to the onset and advancement of bone-related disorders ( Figure 2 ).

Osteoporosis
Osteoporosis is a de gener ative bone disorder marked by reduced bone mineral density (BMD), degraded bone micr ostructur e, incr eased bone fra gility, and a higher susce ptibility to fractures. 198 , 199Fundamentally, it arises from an imbalance where bone resorption surpasses bone formation, resulting in a loss of bone tissue. 200As the population ages, the rates of osteoporosisr elated fra gility fractur es, disa bility, and mortality ar e incr easing year by year. 201umerous studies have confirmed that autophagy increases osteoclast formation under in vitro oxidative stress, low oxygen conditions, and microgravity conditions, leading to an imbalance where bone resorption surpasses bone formation, ultimately triggering osteoporosis ( Figure 3 ). 202 , 203Several studies have demonstrated that miRNAs play a role in regulating bone resorption and bone formation in the process of osteoporosis formation by mediating autopha gy-r elated factors ( Figure 2 ).Resear c h on RAW 264.7 cells under low oxygen conditions r ev ealed that miR-20a dir ectl y targets the 3'UTR of ATG16L1, suppr essing autopha gy by r educing the lev els of autopha gyr elated pr oteins LC-3 and ATG16L1, which is fav ora b le for osteoclast differ entiation.Furthermor e, during osteoclast differ entiation induced by hypoxia, HIF-1 α can regulate miR-20a, and the HIF-1 α-miR20a-ATG16L1 axis plays a significant role. 204MiR-99 ma y pla y a role in fine-tuning and inte gr ating the mTOR signaling pathway to promote optimal osteoclast differentiation. 205 , 206istone demethylases KDM4B and KDM6B play a crucial role in osteogenic commitment of MSCs by removing H3K9me3 and H3K27me3 marks. 207Upregulation of USP7 enhances osteogenic differentiation in human adipose-derived stem cells (hASCs) to suppress osteogenesis progression. 208USP7 promotes the expression of KDM6B by enhancing its stability. 90Elevated levels of miR-15b in osteoporosis inhibit the USP7/KDM6B axis, thereby suppr essing osteob last differ entiation and autopha gy, exacerbating osteoporosis. 209DOT1L, as a histone methyltr ansfer ase , can methylate H3k79.Experiments by Gao et al. confirmed that inhibiting DOT1L in vitr o incr eased autopha gosome assemb l y proteins (ATG3 and ATG8-like Gabarapl2) and autophagy receptors (Sqstm1), acti v ated autopha gy, incr eased pr e-osteoclast migration, and increased bone density.Absorption increases and osteoporosis occurs. 210A resear c h report suggests that ager elated upr egulation of HDAC9 accelerates bone loss in mice by pr omoting dama ged-induced autopha gy. 211Howev er, administering an HDAC9 inhibitor to elderly mice can r estor e mesenchymal osteoblastic function and recover bone mass. 212

Osteoarthritis
Osteoarthritis is a de gener ati v e condition c har acterized by cartilag e deg eneration, subchondral bone remodeling, and synovial inflammation.The development of OA is typically associated with factors such as joint stress, genetic predisposition, obesity, aging, and metabolic or hormonal changes. 213Additionally, inflammatory mediators such as interleukin (IL)-1, IL-6, and TNF ar e excessi v el y pr oduced in chondr ocytes and matrix cells. 214xcessi v e accum ulation of R OS r esulting fr om oxidati v e str ess leads to cartilage de gr adation. 215n the early stages of human OA, the level of autophagy increases, contributing to the maintenance of chondrocyte homeostasis. 216However, in late-stage OA, when chondrocytes endur e pr olonged str ess, autopha gy weakens, leading to further deterioration of cartilage ( Figure 3 ). 217isruption of normal a ge-r elated e pigenetic patterns could contribute to a ge-r elated conditions such as OA. 218Aberrant gene acti v ation in osteoarthritis may be associated with e pigenetic der e pr ession that leads to inflammation and cata bolic meta bolic phenotypes in chondr ocytes.Pr oinflammator y cytokines decr ease methylation at crucial CpG sites in the IL-1 β promoter, resulting in prolonged induction of this cytokine. 219Stimulation of human chondrocytes with IL-1 β leads to decreased methylation at CpG sites in the IL-8 promoter and notably triggers this chemokine in chondrocytes affected by OA. 220 In the study investigating the relationship between autophagy in rat articular chondrocytes and the PI3K/AKT/mTOR signaling pathway in OA, it was found that inflammation can inhibit the proliferation of rat chondrocytes, disrupt the cell cycle, and reduce the rate of autophagy. 221urthermore , DN A demethylation is linked to the upre gulation of essential cartilage-de gr ading proteases such as MMP-3, MMP-9, MMP-13, and ADAMTS-4, as well as iNOS. 222Multiple epigenetic modifications have been found to cooperate in r egulating gene expr ession within the context of osteoarthritic lesions.For example, reduced SOX9 expression in hip OA is likely due to a combination of factors, including ele vated DNA meth ylation, heightened gene-inacti v ating histone marks (H3K9 and H3K27) methylation, and decreased histone acetylation at the SOX9 pr omoter.Furthermor e , miRN A-145 has also been confirmed to inhibit the expression of SOX9 . 223ultiple experiments have also confirmed that miRNAs play a significant role in regulating autophagy in the development of OA ( Figure 2 ).MiR-20 is a member of the miR-17-92 cluster located on the c hromosome . 224In OA, the expression of miR-20 is increased.It can target ATG10 through the PI3K/AKT/mTOR signaling pathway, leading to its reduction, thereby inhibiting chondr ocyte pr oliferation and autopha gy. 225In the cartila ge tissue of OA, miR-375 is found to be ov er expr essed.It can target the ATG2B-3 UTR and inhibit its expression in chondrocytes, suppressing autophagy and promoting endoplasmic reticulum stress (ERs), there by e xacerbating cartilage damage. 226The experiment indicated that in chondrocytes treated with IL-1 β, miR-27a expression is upregulated.It targets the 3 -UTR of the PI3K gene, leading to its downregulation.Through the PI3K-AKT-mTOR pathway, miR-27a activates autophagy and inhibits the prolifer ation of c hondrocytes treated with IL-1 β, providing a protecti v e effect a gainst OA. 227 In addition, m ultiple experiments have confirmed that miR-155, 228 miR-34a, 229 and miR-449a 230 inhibit chondrocyte autophagy, aggravate chondrocyte apoptosis and cause osteoarthritis.

Osteosarcoma
Osteosarcoma is a malignant tumor that originates from mesenc hymal tissue , typically found at the metaphysis of long bones such as the femur, tibia, and humerus.Although it primarily affects children and adolescents, it can also occur in adults. 231he standard treatment for osteosarcoma involves a combination of pr eoperati v e and postoperati v e c hemother apy along with surgical tumor r emov al, which may even necessitate amputation in sev er e cases. 66Despite a ggr essi v e tr eatment, the pr ognosis for osteosarcoma remains relatively poor, with a 5-year survi v al rate of 60%-70% post-surgery.Metastasis, particularly to the lungs, significantly contributes to this unfav ora b le pr ognosis, with r oughl y 20% of patients experiencing metastasis and a subsequent 5-year survi v al rate of only 30%. 232utophagy plays a dual role at different stages of osteosarcoma ( Figure 3 ).In one study, miR-22 was shown to reduce the expression of Beclin1, LC-3, MTDH, and ATG5 mRNA by targeting MTDH, thereby inhibiting autophagy and suppressing osteosarcoma cell pr oliferation. 233Conv ersel y, inhibiting autopha gy w as found to pr omote osteosarcoma cell pr oliferation in another experiment.In this case, miR-19 was upregulated in osteosarcoma cells, targeting SPRED2 and reducing its expr ession, which suppr essed autopha gy, ultimatel y pr omoting the proliferation and malignant transformation of osteosarcoma cells. 234pigenetic regulation plays a significant role in autophagy within the context of osteosarcoma ( Figure 2 ).Trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), induces autophagy in osteosarcoma cells by suppressing the AKT-mTOR signaling pathway and activating FoxO1, thereby enhancing the survi v al of osteosarcoma cells.However, inhibiting autophagy significantly enhances TSA-induced cell death in osteosarcoma. 235Additionally, miR-145-3p, which is downregulated in human osteosarcoma cell lines, targets the 3 -UTR region of HDAC4, increasing HDAC4 levels.This, in turn, promotes apoptosis and autophagy in osteosarcoma cells. 236Another study involving osteosarcoma cells showed that LHX2 ov er expr ession upregulated mTOR expression, which negati v el y r egulated autopha gy thr ough the acti v ation of the mTOR pathw ay, contributing to the pr ogr ession of osteosarcoma.MiR-129-5p dir ectl y targeted LHX2 3 -UTR to downregulate LHX2, making the miR-129-5p/LHX2/mTOR axis a potential target for osteosarcoma tr eatment. 237Additionall y, DANCR is a lncRNA that acts as a ceRNA by sequestering miR-335-5p and miR-1927 in osteosarcoma, pr omoting R OCK1-mediated pr oliferation and metastasis. 238Another study on osteosarcoma r ev eals that miR-193b dir ectl y targets the 3 -UTR of FEN1, negati v el y r egulating the expression of FEN1, increasing the expression of Beclin 1 and the LC3-II/I ratio, acti v ating autopha gy, and inducing cell apoptosis. 239

Rheumatoid Arthritis
1][242] This condition leads to joint deformities and is c har acterized by swelling and pain.As RA pr ogr esses, it can also affect organs and systems outside the joints, such as the heart. 243The treatment of RA typicall y inv olv es a compr ehensi v e appr oach, including medication, ph ysical therap y, and lifestyle management, aimed at alleviating symptoms, controlling inflammation, and maintaining the patient's quality of life. 244Earl y dia gnosis and tr eatment ar e essential for managing the condition and preventing joint damage. 245pigenetic mechanisms, such as DNA demethylation and h ypometh ylation, pla y significant roles in altering DNA methylation patterns during B cell to plasma cell differentiation and in RA.These alterations impact disease pr ogr ession and the expression of key genes. 246 , 247Histone modifications are also inv olv ed in RA, with histone H3 in the promoter region of the IL-6 gene being highly acetylated in fibroblast-like syno vioc ytes (FLS) from RA patients, leading to increased IL-6 expression and disease pr ogr ession.Inhibitors of HAT, suc h as cur cumin, can reduce IL-6 secretion. 248utophagy in RA has a dual role, exerting both positi v e and negati v e effects, de pending on specific cellular and molecular r egulator y mechanisms. 249 , 250Autophagy plays a crucial role in osteoclast differentiation and maturation under hypoxic conditions, leading to increased bone resorption and accelerated progression of RA. 251 Additionally, conditions such as nutrient deficiency and endoplasmic reticulum stress promote autophagy, which acts as a self-pr otecti v e mechanism, allowing RA cells to evade apoptosis and prolong their lifespan. 252esear c h by Li et al. demonstrated that the lncRNA MEG3 is upregulated in synovial tissues of RA patients.MEG3 targets miR-141, and their expression is negatively correlated.By inhibiting the AKT/mTOR pathway and activating autophagy, MEG3 suppresses inflammation, promotes chondrocyte proliferation, and inhibits RA pr ogr ession. 253In experiments by Zhou et al. using a CIA rat model, it was confirmed that treatment with WJR (Wenhua Juanbi Recipe) may inhibit autophagy by affecting the PI3K/AKT/mTOR pathway mediated by miRNA-146a.This inhibition of autophagy leads to the suppression of cell apoptosis and FLS proliferation. 254Moreover, in a study by Yang et al., it was found that ZFAS1 (lncRNA ZNFX1 antisense RNA) plays a r egulator y r ole in FLS-RA thr ough the miR-2682-5p/ADAMTS9 axis.Knockdown of ZFAS1 significantly inhibits FLS-RA cell proliferation, inflammator y r esponse, autopha gy, and pr omotes cell apoptosis ( Figure 2 ). 255

Futur e Dir ections and Challenges
Curr entl y, the gr oup of e pigenetic drugs includes 256 , 257 DNMTis (such as decitabine used in the treatment of AML and high-risk m y elodysplastic syndromes, 258 and 5-azacitidine used in highrisk m y elodysplastic syndromes 259 ), HDAC inhibitors (like TSA mentioned earlier, acti v ating autopha gy in osteosarcoma 235 ), HAT inhibitors (curcumin can reduce IL-6 secretion in RA 248 ), histone methyltr ansfer ase inhibitors (BIX01294 inhibits G9a in multiple m y eloma 260 ), and various miRN A-based molecular targeted therapy drugs.Despite their role in disease treatment, these epigenetic drugs lack specificity in target therapy and often exhibit toxicity.Futur e r esear c h directions in the epigenetic regulation of autophagy for bone meta bolism-r elated diseases aim to minimize these drugs' side effects and identify specific epigenetic modifications involved in autophagy for bone meta bolism-r elated diseases.
The curr entl y well-r esearched histone modifications in bone meta bolism-r elated diseases include histone methylation and acetylation.Other histone modifications, such as histone phosphorylation, 261 ADP-ribosylation, 262 ubiquitination, 263 SUMOylation, 264 glutamylation, 265 glycosylation, 266 hydroxylation, 267 and isomerization, 268 ar e also cov alent modifications that potentially regulate gene expression by altering chromatin structural states and functions, or affecting the affinity between transcription factors and gene promoters.Despite extensi v e resear c h in fields such as tumorigenesis, energy metabolism, and cellular aging, limited reporting exists on their role in bone meta bolism-r elated diseases.Future comprehensive investigations into the regulation of these histone modifications in autophagy concerning bone metabolism-related diseases might un veil ne w targets for pre venting and treating these conditions and guide the development of novel epigenetic drugs.
Resear c h on miRN A re gulation of autophagy in bone meta bolism-r elated diseases is burgeoning and provides potential targets for treating these conditions.A crucial aspect in pr ecise tr eatment for bone-r elated diseases inv olv es transferring target miRNAs or anti-miRNAs to target cells without being de gr aded by endogenous RN A or causing offtarget effects.Pr esentl y, experimental deli v er y systems include aptamers, single-stranded DNA or RNA, 269 , 270 8-r e peat aspartic acid sequences (D-Asp8), 271 and bacteriophage MS2 virus-like particles (MS2 VLPs). 272Futur e adv ancements should emphasize developing delivery systems that enhance miRNA stability and cellular uptake efficiency.MiRNAs, capa b le of mimicking or inhibiting the expression of target genes, have the potential to regulate bone biology processes such as bone formation, resorption, and re gener ation by activating or inhibiting autophagy.

Conclusion
In this paper, we delved into the critical roles of genetic regulation and autophagy in bone metabolism and highlighted key insights r elev ant to bone health.Through the culmination of our resear c h, w e dr aw the following conclusions: Firstl y, genetic r egulation and autopha gy play pi v otal r oles in bone metabolism.Genetic regulation, by modulating gene expression, determines the differentiation, proliferation, and function of bone cells.Autophagy, on the other hand, is a selfr egulating cellular pr ocess indispensa b le for clearing a ged or damaged cells, maintaining intracellular homeostasis, and facilitating bone tissue r e pair.These two mechanisms intricately intertwine and cooperate to uphold bone health.
Secondl y, r esear c h indicates that abnormalities in genetic regulation and autophagy are closely associated with the onset and pr ogr ession of v arious bone meta bolic disorders and bonerelated diseases.Not only do genetic mutations lead to the her editar y transmission of certain bone diseases, but environmental factors, lifestyles, and aging impact the functionality of autophagy, thereby exerting adverse effects on bone health.Consequently, understanding the roles and dysregulation of these r egulator y mechanisms is paramount for pr ev enting, dia gnosing, and treating bone disorders.
Lastly, we emphasize the significance of further resear c h in pr opelling adv ancements in the field of bone health.By delving deeper into the molecular mechanisms of epigenetic regulation and autophagy, we can identify novel therapeutic targets and develop more effective treatment str ate gies.Simultaneously, with the aid of precision medicine and personalized tr eatment appr oac hes, w e can better cater to the di v erse needs of individual patients, elevating the management of bone diseases.
In conclusion, this paper underscores the importance of genetic regulation and autophagy in maintaining bone health and their close connections with bone metabolic disorders and related diseases.We encourage future resear c h to continue delving into these domains, with the aim of providing innov ati v e solutions for pr ev enting and treating bone disorders, ultimately enhancing the quality of life and health of patients.Through collaboration and exploration of new frontiers, we can collecti v el y dri v e pr ogr ess in the field of bone health, paving the w a y for ne w possibilities in future medical and clinical practices.

Figure 1 .
Figure 1.Bone resorption and bone formation maintain the dynamic balance of bone homeostasis.Mesenchymal stem cells in the bone marr ow graduall y differ entiate into osteoblasts, with key transcription factors such as BMPS, Runx2, and OSX playing critical roles in regulating osteoblast differentiation and bone matrix synthesis.Hematopoietic stem cells differentiate into mononucleated osteoclasts under the influence of M-CSF secreted by osteoblasts.Upon activation by RANKL-RANK signaling, these cells further differentiate into mononuclear resorbing cells and subsequently fuse into multinucleated osteoclasts.

Figure 2 .
Figure 2.Under the influence of genetic or pathogenic environmental conditions, histone modifications and ncRNAs promote or inhibit autophagy by targeting autopha gy-r elated genes and proteins.Disordered autophagy leads to the occurrence and progression of bone metabolism-related diseases.restoration of metabolic homeostasis by inhibiting inflammation and excessi v e autopha gy.

Figure 3 .
Figure 3. Epigenetic modifications r egulate autopha gy in bone metabolic diseases.(A) Histone modification and miRNA's inhibition of autophagy results in enhanced osteoclast generation and impedes osteoblast differentiation, causing increased bone resorption relative to bone formation, ultimately leading to osteoporosis.(B) DNA methylation modification and miRNA's inhibition of autophagy lead to incr eased chondr ocyte apoptosis, hinder ed chondr ocyte pr oliferation, and differ entiation, leading to the onset of osteoarthritis.(C-F), Autophagy demonstrates a dual role in osteosarcoma.Its inhibition can either promote or impede the proliferation of osteosarcoma cells.Sim ultaneousl y, inducing autophagy can trigger cell apoptosis or elevate the survival rate of osteosarcoma cells.

Table 1 .
Histone acetyltransferase, histone deacetylase, and their role in bone metabolism.

Table 2 .
Common histone methyltransferases and demethylases inv olv ed in histone methylation modification, as well as their target sites, genes, and their roles in bone metabolism. 82 Ta ble 3. LncRN A participates in bone metabolism by functioning as a miRNA sponge or by modulating the activity of transcription factors and signaling pathways.

Table 4 .
The role of miRNA in osteogenesis and bone cell differentiation.