Autophagy plays an essential role in bone homeostasis

Abstract

Autophagy is very critical for multiple cellular processes. Autophagy plays a critical role in bone cell differentiation and function.

1. INTRODUCTION

Autophagy, a major catabolic process in eukaryotic cells is evolved for the degradation and recycling of damaged organelles and macromolecules. Growing body of evidences suggest the role of autophagy in skeletal homeostasis, in particular the remodeling of bone as evidence by autophagic response of bone cells‐osteoclasts, osteoblasts and osteocytes. Deficiency in autophagy may results into several pathologies such as osteoporosis and sarcopenia. The aim of this review is to briefly highlight the recent advances in the involvement of bone cells exhibiting autophagy and provide evidence that autophagic dysfunction in these cells results in the pathogenesis of bone diseases. This review further summarizes the role of autophagy in the regulation of differentiation of bone cells. Moreover, role of autophagy in the regulation of skeletal homeostasis in context of aging was described. These recent development also shed new light on the essential roles of autophagy in the regulation of differentiation and function of osteoclasts, osteoblasts and osteocytes. Bone is a specialized connective tissue and the main component of the adult skeleton that provides mechanical support for the body protection of vital organs and regulates mineral homeostasis (Mescher & Junqueira, 2013). Bone contains three main cell types: osteoblasts, osteocytes, and osteoclasts. These cells represent approximately 15% of the total bone weight (Alberts, Johnson, Lewis, & Morgan, 2015) and are responsible for growing and maintaining homeostasis and healthy bone. Osteoblasts lie on the surface of the bone matrix and have a cuboidal or columnar shape in addition to a basophilic cytoplasm (Mescher & Junqueira, 2013). These cells differentiate from mesenchymal stem cells in bone marrow (Alberts et al., 2015). The most important factor that is responsible for osteoblast differentiation from mesenchymal stem cells is Runt related transcription factor 2 (RUNX2) (Abdallah, Jafari, Zaher, Qiu, & Kassem, 2015), a transcription factor that increases the expression of osteoblast‐related markers when bound in the proximal promoter region, but acts as an inhibitor of osteoblastogenesis when bound in the distal promoter region. RUNX2 is essential for both intramembranous and endochondral ossification (Bruderer, Richards, Alini, & Stoddart, 2014). Another transcription factor stimulated by p38 mitogen‐activated protein kinase is osterix, which is important for osteoblast differentiation. Bone morphogenetic proteins and transforming growth factor‐β are also important factors for osteoblastogenesis and signaling through Smad signaling pathways (Abdallah et al., 2015). The Wnts/β‐catenin pathway also plays a critical role in bone development (Day, Guo, Garrett‐Beal, & Yang, 2005). The Wnts signaling family acts through different signaling pathways. It is expressed by osteoblasts in bone marrow and increased bone mass (Andrade, Nilsson, Barnes, & Baron, 2007). Peroxisome proliferator‐activated receptor γ (PPARγ) is an inhibitory transcription factor for osteoblast differentiation that works by suppressing RUNX2 expression and activity (Colaianni, Brunetti, Faienza, Colucci, & Grano, 2014). PPARγ has a relationship with β‐catenin, which inhibits osteoblastogenesis and stimulates the adipogenesis pathway (Colaianni et al., 2014; Figure 1). Taken the above together, it can be said that multiple pathways play a crucial role in osteoblast differentiation and function.

Figure 1.

Schematic diagram illustrates the osteoblast that arises from mesenchymal stem cells (MSCs) and requires transcription factors, Runx2 and osterix, to differentiate into osteoblast. The osteoblast in culture undergoes three distinct stages (proliferations from Day 0 to Day 7, differentiation from Day 7 to Day 14, and mineralization from Day 14 to Day 21). Alkaline phosphatase (ALP) is the early marker; bone sialoprotein and osteocalcin are the late markers for osteoblast differentiation. Osteoblast may undergo apoptosis, become an osteocyte, or lie flat on the bone surface and become a bone lining cell. Mesenchymal stem cells can also differentiate into myoblast, chondrocyte, and adipocyte [Color figure can be viewed at wileyonlinelibrary.com]

Osteocytes are located in cavities called lacunae, which lie within the bone matrix. The material secreted by differentiated osteocytes gradually surrounds osteoblasts. Osteocytes are flat in shape and maintain the bone matrix and die after matrix resorption (Mescher & Junqueira, 2013). Osteocytes can live many months or years depending on the location and rate of bone remodeling in contrast to osteoblasts and osteoclasts, which have a short lifespan (Manolagas & Parfitt, 2010). In addition, osteocytes are the main source of receptor activator of nuclear factor kappa‐light‐chain‐enhancer of activated B (NFkB) ligand (RANKL) that regulates osteoclast differentiation (Nakashima et al., 2011; Xiong et al., 2011); thus, the low bone mass may be attributed to a change in osteocyte function (Manolagas & Parfitt, 2010).

Osteoclasts are large and multinucleated cells that play a critical role in bone resorption during bone growth and remodeling. Macrophage‐colony‐stimulating factor (M‐CSF) and RANKL are required for osteoclast development (Mescher & Junqueira, 2013) to redirect hematopoietic stem cell (HSC) differentiation into mature osteoclasts (Väänänen & Laitala‐Leinonen, 2008), and are also crucial for recruiting osteoclasts to sites undergoing bone resorption (Alberts et al., 2015). RANKL1 and RANKL2 are the transmembrane isoforms of RANKL that are important for osteoclast differentiation and function by binding the RANK receptor (RANK) (Theoleyre et al., 2004). Another isoform is RANKL3, which is a soluble form and is responsible for inhibiting the other two forms when coexpressed with RANKL1 and RANKL2 (Theoleyre et al., 2004). Another factor that plays a role in osteoclast differentiation is osteoprotegerin, which blocks RANK/RANKL binding and, thus, inhibits osteoclast differentiation (Marieb & Hoehn, 2016). Other factors are also important for osteoclastogenesis, such as nuclear factor of activated T cells (NFAc1; T. Wada, Nakashima, Hiroshi, & Penninger, 2006). The last step of osteoclast differentiation is cell fusion. This process occurs mainly through two different types of cell contacts: phagocytic‐cup and broad contact surfaces. More mature multinucleated osteoclasts fuse with a less mature mononucleated preosteoclasts. One important factor for osteoclast fusion is dendritic cell‐specific transmembrane protein that is found on the surface in preosteoclasts (Søe, Hobolt‐Pedersen, & Delaisse, 2015). After differentiation, osteoclasts attach to the mineralized bone matrices through dot‐like, actin‐rich structures called podosomes. Podosomes are interconnected by a circular “actin ring” comprised of F‐actin, which creates a sealed area for bone resorption. The podosomes rely on the CD44 receptor and αvβ3 integrin for function; both are important to start attachment to the bone surface (Figure 2; Holt & Marshall, 1998; Nakamura, Gailit, & Sasaki, 1996; Väänänen & Laitala‐Leinonen, 2008).

Figure 2.

Schematic diagram of osteoclast differentiation from hematopoietic stem cells (HScs). Myeloid precursor starts at Day 0 to proliferate and differentiate to osteoclast. By Day 3 in culture, bone‐marrow macrophages (BMM) express high levels of MCSFR and RANK that stimulate osteoclast precursor cells differentiation. By Day 7 in culture, preostoeclasts become mature osteoclast. Cathepsin K is an osteoclast marker. DC‐STAMP, dendritic cell‐specific transmembrane protein; MCSFR: macrophage‐colony‐stimulating factor; RANK: receptor activator of NFkB [Color figure can be viewed at wileyonlinelibrary.com]

Osteopontin, an example of many regulators of bone remodeling, is an extracellular matrix protein, along with integrins, which connects osteoclast to bone (Z. Li, Kong, & Qi, 2006). Proteins that contain the arginine‐glycine‐aspartic acid peptides help osteoclast attachment to the surface and bone resorption, and they can reorganize the osteoclast cytoskeleton (Vaananen, 2005). An important factor that plays a role in osteoclast differentiation and bone resorption is tartrate‐resistant acid phosphatase (TRACP), a biomarker for osteoclast activity that is stimulated by cathepsin‐K enzyme. In addition, reactive oxygen species, generated by TRACP activity, play a role in the degradation of the collagen matrix (Väänänen & Laitala‐Leinonen, 2008). Deficiency in TRACP leads to an osteopetrotic phenotype and decreases mineralization of bone development in mice (Hayman et al., 1996).

Osteoclasts express high levels of calcitonin receptor (CTR), which inhibits osteoclast resorption. CTR is considered a marker for osteoclast differentiation (S. Wada, Martin, & Findlay, 1995). Changes in the intracellular level of calcium affects bone resorption (Adebanjo, Moonga, Haddad, Huang, & Zaidi, 1998), and it has been shown that increased calcium levels can induce apoptosis in osteoclasts (Lorget et al., 2000) due to an increase in membrane permeability to protons and other ions (Lehenkari et al., 2003). At the end stage of bone resorption, osteoclasts die by apoptosis (Kameda, Ishikawa, & Tsutsui, 1995).

2. INTERACTION BETWEEN OSTEOBLAST AND OSTEOCLAST

Osteoblasts and osteoclast communicate by paracrine signaling through cytokines and gap junctions; both cells are located on the bone surface (Matsuo & Irie, 2008). Osteoblasts migrate to resorption pits for laying down fresh bone matrix (Henriksen, Neutzsky‐Wulff, Bonewald, & Karsdal, 2009). High levels of TRACP in the resorption pits appear to stimulate osteoblast differentiation (Henriksen et al., 2009). Additionally, mononuclear cells are necessary for preparing the resorption pits for the fresh bone matrix (Henriksen et al., 2009). Taken together, osteoclasts stimulate osteoblast differentiation and formation of new bone during the remolding (Henriksen et al., 2009). Osteoblast function and osteoclast number also appear to be linked, because the number of osteoclasts controls bone formation independent of their resorption activity (Henriksen et al., 2009).

3. AUTOPHAGY

The term autophagy is derived from Greek and refers to a self‐eating process that removes misfolded proteins, clears damaged organelles, and eliminates intracellular pathogens (Glick, Barth, & Macleod, 2010). Autophagy is also important to balance the energy sources during nutrient stress and starvation. Autophagy also plays a role in preventing numerous diseases, such as autoimmune diseases, diabetes, and infections (Glick et al., 2010). A deficiency in autophagy contributes to Parkinsons’s disease (Arduíno, Esteves, & Cardoso, 2013), neurodegeneration, and cancer (Kundu & Thompson, 2008; Levine & Kroemer, 2008). Autophagy is the most important and necessary pathway regulating cell growth, differentiation, and function (Awan & Deng, 2014; Lamy et al., 2013). Autophagy and apoptosis (programmed cell death) are related to each other; autophagy provides a basis for apoptosis, and inhibition of autophagy leads to delayed apoptosis (Wu et al., 2014). Autophagy maintains cell balance by recycling damaged material to reuse these cytosolic components (Awan & Deng, 2014). There are three types of autophagy: microautophagy, chaperone‐mediated autophagy, and macroautophagy. Microautophagy in mammalian cells is defined as a nonselective lysosomal degradation involved in long‐lived protein turnover. Soluble‐substrate microautophagy can be stimulated by nitrogen starvation or by rapamycin, leading to high microtubule‐associated protein light chain 3 (LC3) levels (Mijaljica, Prescott, & Devenish, 2011). Chaperone‐mediated autophagy is specialized for the degradation of soluble cytosolic proteins. Chaperone proteins translocate the targeted proteins across the lysosomal membrane. This type of autophagy can be triggered by stress such as oxidative stress and toxic material exposure (Kaushik & Cuervo, 2012). Macroautophagy regulates the arrangement of cellular components that have to be recycled via the lysosome (Kanazawa et al., 2004; C. W. Wang & Klionsky, 2003). In mammalian cells, macroautophagy is stimulated by decreased levels of insulin, and it delivers the cytoplasmic components to lysosomes through structures called autophagosomes (Mortimore, Reeta pösö, & Lardeux, 1989).

Autophagy has various steps to remove unwanted cellular components (Xie & Klionsky, 2007). The initiation of autophagy starts with the formation of a membrane known as the phagophore; this is derived from the lipid bilayer membrane taken from the endoplasmic reticulum or trans‐Golgi (Simonsen & Tooze, 2009). Both ends of the phagophore membrane elongate to engulf the targeted components and form an autophagosome structure. Autophagosome maturity occurs through fusion with the lysosome, which contains lysosomal acid proteases that degrade the engulfed materials(Figure 3; Awan & Deng, 2014; Glick et al., 2010; Jiang et al., 2014; Mizushima, 2007; Mizushima, Yoshimori, & Ohsumi, 2011).

Figure 3.

Schematic diagram of the steps of autophagy. (a) Autophagy begins with isolation of the membrane called phagophore. Collection of cellular components form autophagosome. Autophagosome fuses with the lysosome to form autolysosome. The membrane is lysed and autophagic cargo is digested by lysosomal proteases. (b) Inhibitors of autophagy process at any step as indicated here. 3‐Methyladenine blocks the early step of autophagy (membrane formation). Bafilomycin A1 is responsible for blocking fusion between autophagosome and lysosome, and chloroquine blocks the last step autolysosome. LC3: light chain 3 [Color figure can be viewed at wileyonlinelibrary.com]

4. REGULATION OF AUTOPHAGY

Macroautophagy is regulated by more than 30 autophagy‐related genes (ATG) that have been identified in yeast and mammals; most have a role in autophagosome membrane formation (Reggiori & Klionsky, 2002; Yu et al., 2004). ATG1 is the key protein initiating autophagy by forming a complex with ATG13 and ATG17, both of which increase the kinase activity of ATG1 to activate the formation of the autophagosome. ATG13 stimulates the interaction of ATG1–ATG1, which correlates with the kinase activity of ATG1 to promote autophagy (Yeh, Shah, & Herman, 2011). UNC‐51‐like kinase (ULK1), a major negative regulator of autophagy (ATG13), and family interaction protein of 200 KD (FIP200) are the mammalian homologs of the yeast ATG1, ATG13, and ATG17, respectively (Hara & Mizushima, 2009; Hosokawa et al., 2009). Microtubule‐associated protein LC3, the mammalian homolog of ATG8, plays a role in the elongation process during autophagosome formation, and binds to ATG1, increasing its activity (Kraft et al., 2012). Beclin‐1, the mammalian homolog of yeast ATG6, is the molecule most directly associated with autophagy and is required for the initial step of autophagosome formation. Beclin‐1 and class III phosphatidylinositol 3‐kinase (III PI3K) form a complex that also includes vesicular protein sorting 34 (Vps34) and p150 (Simonsen & Tooze, 2009). Other regulators for autophagy include mitogen‐activated kinases (BNIP3), which is a member of the Bcl‐2 family (Vande Velde et al., 2000), AMP‐activated protein kinase, and calcium. Tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand is also essential to induce autophagy during formation of lumen in vitro (Table 1; Mills, Reginato, Debnath, Queenan, & Brugge, 2004).

Table 1.

Autophagy‐related genes and their function

Name of gene	Role of autophagy	Reference
ATG1	Induction of autophagy	Reggiori and Klionsky (2002)
ATG5	Autophagosome formation	Reggiori and Klionsky (2002)
ATG7	Autophagosome formation	Reggiori and Klionsky (2002) and Yu et al. (2004)
ATG8	Marker of autophagosome formation	Reggiori and Klionsky (2002)
ATG12	Autophagosome formation	Reggiori and Klionsky (2002)
ATG13	Induction of autophagy	Reggiori and Klionsky (2002)
Beclin ‐1	Initiation of autophagosome formation	Simonsen and Tooze (2009)

During starvation, proteins are broken down by autophagy to release amino acids and increase ATP levels (Kanazawa et al., 2004; Mortimore et al., 1989). Macroautophagy degrades lipid droplets into free fatty acids (lipophagy) (Singh et al., 2009), while macroautophagy and microautophagy break down glycogen into oligosaccharides and glucose (glycophagy) (Kotoulas, Kalamidas, & Kondomerkos, 2006). Defective autophagy results in disrupted metabolic homeostasis. For example, defects in lipid degradation lead to toxic accumulation of triglycerides (Singh et al., 2009), and defective glycophagy leads to glycogen deposition in the cytosol (Kotoulas et al., 2006). Autophagy allows maintenance of energy homeostasis during neonatal starvation (Kuma et al., 2004). Moreover, mice lacking ATG5 display defective engulfment of the apoptotic corpse during development (Qu et al., 2007). This suggests that autophagy contributes to dead‐cell clearance during programmed cell death by the generation of energy. Autophagy can inhibit human malignancies and its level is decreased in prostate cancer (Jiang et al., 2014). Knockout autophagy genes in animals in the central nervous system lead to neurodegeneration in mice due to the accumulation of cytoplasmic inclusion bodies in neurons and cause deficits in motor function (Hara et al., 2006; Komatsu et al., 2006). Autophagy is needed for preimplantation of embryos for protein recycling; inhibition of autophagy in oocytes obtained from oocyte‐specific ATG5 knockout mice delays the development of mouse embryos compared with wild‐type mice (Tsukamoto et al., 2008). In addition, autophagy proteins are important for plant cells, for the development of a root system under nutrient starvation (Yoshimoto et al., 2004). In addition to nutrient starvation, autophagy might be responsive to hypoxic conditions (Adhami et al., 2006), such as ischemic myocardium (Matsui et al., 2007; Yan et al., 2005). This is mediated by hypoxia‐inducible factor 1 (HIF‐1), which is the main regulator of hypoxic responses (Bohensky et al., 2007).

5. AUTOPHAGY AND BONE HOMEOSTASIS

Autophagy has a role in bone homeostasis because of the large amounts of waste materials that are generated during bone resorption. Osteoblasts and osteoclasts are bone cells with opposing roles; osteoblasts build bone whereas osteoclasts resorb bone. Imbalances between bone formation and resorption result in skeletal disorders including osteoporosis, osteopetrosis, and Paget’s disease of bone (PDB). Autophagy‐related proteins have been shown to be important for cartilage repair and are mediators in bone cell differentiation and function, suggesting that autophagy plays a role in bone homeostasis (Hocking, Whitehouse, & Helfrich, 2012). Loss of autophagy could lead to bone cell dysfunction, because it is known that during differentiation, osteoblasts use autophagic vacuoles as a source of energy (Nuschke et al., 2014) in addition to the role of autophagy on osteoclast function (DeSelm et al., 2011).

6. The ROLE OF AUTOPHAGY IN OSTEOBLAST DIFFERENTIATION

Osteoblasts are the cells responsible for the synthesis of bone matrix and mineralization. The neighbor of BRCA1 gene (NBR1) is an autophagy receptor that interacts with LC3 and with ubiquitinated, aggregated proteins to degrade the ubiquitinated substrates. Disruption of NBR1 has a significant positive effect on osteoblast differentiation and function (Kirkin et al., 2009; Waters, Marchbank, Solomon, Whitehouse, & Gautel, 2009). Genetic truncation of murine Nbr1 enhances osteoblast differentiation and activity, which leads to an age‐dependent increase in bone mass and density (Whitehouse et al., 2010). Decreased levels of autophagy in human mesenchymal stem cells lead to reduced differentiation into osteoblasts (Oliver, Hue, Priault, & Vallette, 2012). Another study showed that a target deletion of the FIP200, part of the complex that initiates autophagosome formation, leads to a reduction in bone formation (Y. Liu et al., 2013). A separate study, which used a high‐throughput small‐molecule screening assay, found that rapamycin induced autophagy in osteoblast cells and bafilomycin A1 caused accumulation of autophagic structures (Oliver et al., 2012). In summary, these studies suggest that autophagy enhances bone formation. By using osteoblast cell lines, a study showed that autophagy is enhanced during osteoblast differentiation and mineralization (Y. Liu et al., 2013; Nollet et al., 2014). Double‐membraned autophagic vesicles in the osteoblast cells contained structures that released apatite crystals in the extracellular medium (Nollet et al., 2014). The role of autophagy in osteoblast function was confirmed by inhibiting autophagy using 3‐methyladenine or chloroquine on Day 10 of osteoblast differentiation and Day 21 of osteoblast mineralization, which led to decreased alkaline phosphatase on Day 10 and a reduction in bone mineralization on Day 21 (F. Liu et al., 2013). Other autophagy inhibitors, including bafilomycin, chloroquine, ammonium chloride (NH₄CL), or short hairpin RNA‐mediated knockdown of the autophagy‐essential gene LC3‐β inhibit osteogenic differentiation in human dental pulp mesenchymal stem cells (Pantovic et al., 2013). Treatment with small interfering RNA for ATG7 or BCN1 (siATG7 and siBCN1) has been shown to decrease mineralization in a rat osteoblast cell line (Table 2 and Figure 4; Nollet et al., 2014).

Table 2.

Autophagy‐related genes regulate bone cells

Osteoblast
Name	Function	References
NBR1	Increase osteoblast differentiation and activity Increase bone mass and density.	Whitehouse et al. (2010)
Deletion of Fip200	Reduction in bone formation.	Y. Liu et al., 2013
Rapamycin	Increased osteoblast differentiation.	Oliver et al. (2012)
3‐MA and chloroquine	Decreased number and size of alkaline phosphatase at Day 10 Reduction in bone mineralization at Day 21.	F. Liu et al. (2013)
Bafilomycin, chloroquine, NH4CL, and shRNA‐mediated knockdown of LC3‐B	Blocked osteogenic	Pantovic et al. (2013)
siATG7 and siBCN1	Decreased osteoblast mineralization.	Nollet et al. (2014)
Osteoclasts
Mutation in SQSTM1 (P62)	Paget’s disease	Helfrich and Hocking (2008)
ATG5, ATG7, ATG4B, and LC3	Generating the osteoclast‐ruffled border.	DeSelm et al. (2011)
Deletion of ATG5 and ATG7	Reduce the restorative capacity of osteoclast.	DeSelm et al. (2011)
Hypoxia	Increased osteoclast size and differentiation.	Arnett (2010), Bozec et al. (2008), Sambandam et al. (2014), and Y. Zhao et al. (2012)
Absent of ATG7	HSCs fail to differentiate into osteoclast	Mortensen et al. (2011)
P62	Increased osteoclastogenesis.	R. F. Li et al. (2014)
Decrease level of Beclin‐1	Reduction in osteoclast differentiation and function.	Chung et al. (2014)

Note. 3‐MA: 3‐Methyladenine; shRNA: short hairpin RNA.

Figure 4.

Schematic diagram illustrates the role of autophagy on osteoblast differentiation and bone formation [Color figure can be viewed at wileyonlinelibrary.com]

7. The ROLE OF AUTOPHAGY IN OSTEOCYTE DIFFERENTIATION

Osteocytes are terminally differentiated cells that are formed from osteoblasts, located in mineralized bone matrix transition. During this transition, cells undergo a change in shape and a decrease in organelle contents and size (Zahm, Bohensky, Adams, Shapiro, & Srinivas, 2011; Marotti, Zallone, & Ledda, 1976). An increase in autophagy may accompany this transition to assist in adjusting organelle size and to provide raw materials for protein and the membrane as the osteocytes acclimate to the hypoxia and poor nutrient conditions within the matrix. In support of this notion, another study showed a higher expression of LC3 in osteocytes compared to cell surface osteoblasts in rat tibia (Zahm et al., 2011). Furthermore, in vitro studies showed that the more differentiated osteocytes have higher autophagic flux (Zahm et al., 2011). Another study showed that deletion of ATG7 from osteocytes results in a significant reduction of bone mass (H. X. J. Zhao & Onal, 2012). Treatment with glucocorticoids also led to autophagy induction that saved osteocyte viability (Jia et al., 2011; Xia et al., 2010). Inhibition of autophagy does not have an effect on glucocorticoid in the bone (Piemontese et al., 2015). These data suggest that autophagy plays an important role in osteocyte survival, which, in turn, protects bone from loss.

8. THE ROLE OF AUTOPHAGY IN OSTEOCLAST DIFFERENTIATION

Bone resorption is performed by osteoclasts, which are multinucleated cells derived from HSCs after induction by M‐CSF and RANK ligand. Autophagy has a role in the regulation of osteoclast function. Studies found that PDB, which is characterized by increased activity, size, multinuclearity, and a number of osteoclasts can occur as a result of a mutation in the autophagic cargo receptor Sequestosome‐1 (SQSTM1), also known as the ubiquitin‐binding protein p62 (P62) (Helfrich & Hocking, 2008). Osteoclasts resorb bone by the ruffled border. Lysosomal fusion with the plasmalemma results in the release of Cathepsin K (CatK) to resorb bone matrix. ATG5, ATG7, ATG4β, and LC3 are the main proteins for autophagosome formation generating the osteoclast‐ruffled border in vivo and in vitro (DeSelm et al., 2011). Deletion of ATG5 and ATG7 reduced the resorptive capacity of osteoclasts without affecting their differentiation (DeSelm et al., 2011). Furthermore, other studies confirmed that hypoxia causes formation of multinucleated osteoclasts in young pups during embryogenesis (Arnett, 2010; Bozec et al., 2008). Another study showed that HIF‐1 alpha and microgravity caused increased osteoclast differentiation and a significant increase in autophagy (Sambandam et al., 2014; Y. Zhao et al., 2012). Induced autophagy by rapamycin led to a decrease in osteoclast number in rats (Sanchez & He, 2009), a reduction in osteoclast differentiation and bone resorption in mouse models of arthritis (Cejka et al., 2010), decreased bone resorption in patients with renal transplantation (Westenfeld et al., 2011), and limited in vitro osteoclast formation in patients with giant cell tumor (Smink, Tunn, & Leutz, 2012). Autophagy plays an essential role in the maintenance of HSC. HSCs fail to differentiate into osteoclasts when ATG7 is absent (Mortensen et al., 2011). P62 adaptor protein plays a critical role in RANKL promoted autophagy and osteoclastogenesis (R. F. Li et al., 2014).

Recently, it has been reported that decreased levels of Beclin‐1 in osteoclast led to a reduction in differentiation and function of these cells, working through NFATc1 (Chung et al., 2014). Increased expression of Beclin‐1 and ATG7 in human rheumatoid arthritis accompanied TNF‐activated autophagy in vivo and in vitro that regulated osteoclast differentiation and bone resorption (Lin et al., 2013). Autophagy mediates fusion between lysosomal contents and the plasma membrane by participating in polarized secretion into the extracellular space when lysosomes fuse with the plasma membrane (DeSelm et al., 2011). ATG7 knockdown inhibits expression of osteoclast markers TRAP and CatK during osteoclast differentiation (K. Wang, Niu, Kim, & Kolattukudy, 2011). The lysosome fusion with the ruffled border led to protease secretion into the resorptive lacunae (Itzstein, Coxon, & Rogers, 2011), and induced autophagy led to reduced pathological osteoclast formation (Table 2 and Figure 5; Smink et al., 2012).

Figure 5.

Schematic diagram illustrates the role of autophagy on osteoclast differentiation and bone resorption. RANKL: receptor activator of NFkB ligand [Color figure can be viewed at wileyonlinelibrary.com]

9. The ROLE OF AUTOPHAGY IN AGING BONE

Mutation or knockout of autophagy components in bone leads to age‐dependent phenotypes (Daroszewska et al., 2011; DeSelm et al., 2011; Hocking et al., 2004). For example, ATG5 knockout mice have higher trabecular bone volume compared with the wild‐type 8‐month‐old mice (DeSelm et al., 2011; Xia et al., 2010). Autophagy decreases with age, and this is clear in the terminally differentiated cells (Hubbard, Valdor, Macian, & Cuervo, 2012). The decreased autophagy in humans has been observed to be involved in many neurodegenerative diseases. Increased autophagy weakens the features of neurodegeneration. This effect has been associated with some disorders such as Hutchinson–Gilford progeria syndrome, a deadly genetic disorder categorized by premature aging (Cao et al., 2011). Inclusion body myopathy (IBM) linked with PDB and frontotemporal dementia, also known as IBMPFD, is considered one of the diseases that are associated with aging and the abnormal function of autophagy (Watts et al., 2004). PDB is known to be a common disorder, which affects approximately 3.1% of people over 55 years old in the United States (Hocking et al., 2004). One of the most significant roles that autophagy plays has to be associated with the existence and importance of proteins, which include ATG5, ATG7, ATG4B, and LC3, considering how it is needed in producing the osteoclast‐ruffled borders, which are essential in keeping with a reputed connection between human skeletal homeostasis and autophagy genes (DeSelm et al., 2011). This effect is more pronounced in terminally differentiated bone cells (Cuervo et al., 2005). Other studies have focused on developing an in vivo assessment to quantify the bone forming capacity (BFC) and have also compared the BFC of osteoblastic cells gained from young and old donors (Stenderup et al., 2004). Moreover, other researchers focused on identifying a possible solution, such as how caloric restriction protects against disease and even aging (Selman et al., 2009).

10. CONCLUSION

The existence of autophagy has already been known to play a very significant role in tissue homeostasis and physiology. The existing data provide clarification for essential roles of autophagy in regulating differentiation and function of bone cells. In addition, autophagy deficiency leads to some bone pathologies. Taken together, much work is still needed to examine the physiological and therapeutic roles of autophagy for bone diseases.

Jaber FA, Khan NM, Ansari MY, Al‐Adlaan AA, Hussein NJ, Safadi FF. Autophagy plays an essential role in bone homeostasis. J Cell Physiol. 2019;234:12105–12115. 10.1002/jcp.27071