Increase, Cell death leads to Altered Bone Cell Homeostasis

The biological rationale for the connection of cell death and altered bone cell homeostasis is well-supported in the literature. Bone homeostasis is regulated by the balanced action of bone-forming osteoblasts and bone-resorbing osteoclasts and by the action of osteocytes, the “mechano-sensing cells” in the compact bone. Research has shown that osteocyte apoptosis-induced bone resorption plays a role in regulating bone homeostasis/bone mass (Komori, 2013). Briefly, apoptotic osteocytes release of osteoclast stimulatory factors that recruit pre-/osteoclasts locally to the apoptotic cell (Jilka, Noble, and Weinstein, 2013; Komori, 2013; O’Brien, Nakashima, and Takayanagi, 2013; Plotkin, 2014; Xiong and O’Brien, 2012). Further osteoblast death may impair bone formation as the pool of active bone-forming osteoblasts decreases. 

Regardless of if cells undergo apoptosis or autophagy, death is completed with the removal of the cells through engulfment by scavengers. In these cases, the cells are quietly removed without inflammation, because the integrity of the cytoplasmic membranes is maintained when phagocytosis occurs. In the case of apoptotic osteocytes, scavengers cannot reach osteocytes that are embedded in the compact bone and, thus, any type of osteocyte death will end in the rupture of the cytoplasmic membrane (Komori, 2013). After cell rupture, immunostimulatory factors are released to the bone surface and vascular channels and facilitate the recruitment and activation of macrophages, thereby promoting the production of proinflammatory cytokines that in turn facilitates osteoclastogenesis and bone resorption (Komori, 2013). The relationship between osteocyte apoptosis and increased local bone resorption has been verified by studies showing co-localization of apoptotic osteocytes and recruited osteoclasts, blockade of osteocyte apoptosis reduced bone resorption, and osteocyte apoptosis preceding osteoclast recruitment (Jilka, Noble, and Weinstein, 2013; O’Brien, Nakashima, and Takayanagi, 2013; Plotkin, 2014; Xiong et al., 2013). However, the exact mechanism how apoptotic osteocytes recruit osteoclasts is still debated.  

It has been shown that after rupture of the plasma membrane of dead osteocytes immunostimulatory factors such as high-mobility group box 1 (HMGB1) are released, facilitating the recruitment and activation of macrophages, thereby promoting the production of proinflammatory cytokines such as TNF-α, IL-6 and IL-1. IL-6 and IL-1 induce RANKL expression, that in turn facilitates osteoclastogenesis and bone resorption (Jilka, Noble, and Weinstein, 2013; Komori, 2013). Other studies, however, propose that apoptotic osteocytes signal to viable osteocytes in their vicinity to express high ratios of RANKL/OPG (RANKL being the main stimulator of osteoclastogenesis and OPG, osteoprotegerin, its inhibitor) and other pro-osteoclastogenic factors that directly stimulate osteoclast recruitment and enhance the production of mature osteoclasts (O’Brien, Nakashima, and Takayanagi, 2013; Plotkin, 2014).  

Autophagy is part of the regulation process of osteoclast differentiation and function and thus linked to bone resorption. Regarding bone resorption, osteoclasts encounter a low oxygen tension in their local environment as they are living at the surface and interior parts of the bone (Shapiro et al., 2014). Different studies have reported that hypoxia via activation of HIF-1α (hypoxia inducing factor-1α) enhances osteoclast differentiation and activity along with autophagic flux (Knowles and Athanasou, 2009). HIF-1α induces the expression of its downstream target BNIP3, which stimulates Beclin-1 release, increases the expression level of autophagic-related genes such as ATG5 and ATG12, recruits LC3 to autophagosome, and enhances the expression of osteoclast genes (nuclear factor of activated T cells 1 (NFATc1), tartrate-resistant acid phosphatase (TRAP), Cathepsin K (CTSK), and matrix-metalloproteinases (MMPs)) (Zhao et al., 2012). It also has been shown that upon activation of the osteoclast receptor RANK, by osteoblast-secreted and osteocyte-secreted RANKL, leads to the recruitment of TRAF6 and an increase of Beclin-1 and ATG5/7/12 with enhanced activation of LC3. Further, formed autophagosomes and lysosomes are directed to the ruffled border where bone resorption takes place (Chatziravdeli et al., 2019; Lacombe, Karsenty, and Ferron, 2013). 

The empirical data obtained for this KER strongly supports a link between apoptosis and altered bone cell homeostasis. This evidence comes from studies examining the effects of microgravity exposure and various forms of ionizing radiation, including gamma rays and X-rays, which directly induced apoptosis of bone cells and resulted in a dose-dependent increase in bone resorption and a dose-dependent decrease in bone formation (Aguirre et al., 2009; Chandra et al., 2017; Chandra et al., 2014; Huang et al., 2019; Huang et al., 2018; Li et al., 2020; Li et al., 2015; Liu et al., 2018; Wright et al., 2015).  

Incidence concordance 

There is some evidence that cell death increases more than bone cell homeostasis is altered following a stressor. In vivo osteoblast apoptosis in rats increased 7- fold while osteoblast numbers decreased 0.25-fold after irradiation with 8 Gy of X-rays (Chandra et al., 2014). Similarly, mice irradiated with 8 Gy of X-rays showed a 4-fold increase in osteoblast apoptosis and a 0.5-fold decrease in osteoblast number (Chandra et al., 2017). In vitro, human bone marrow-derived mesenchymal stem cells (hBMMSCs, osteoblast precursors) irradiated with 8 Gy of X-rays showed a 3-fold increase in apoptosis and osteoblasts subsequently had a 0.5-fold decrease in alkaline phosphatase (ALP) activity (Liu et al., 2018). Huang et al. (2019) showed very similar results in rats with a 4-fold increase in osteoblast apoptosis and a 0.3-fold decrease in ALP activity after irradiation with 2 Gy of gamma rays. Osteoblast irradiated with gamma rays at 10 Gy showed a 3-fold increase in caspase-3 and a 0.7-fold decrease in ALP activity (Li et al., 2015). 

Dose Concordance 

Current literature provides evidence suggesting a dose concordance relationship between cell death of bone cells and altered bone cell homeostasis. Studies examining the effects of microgravity exposure on osteocytes in vivo have found a significantly increased number of empty lacunae suggesting significantly enhanced osteocyte apoptosis; which coincided with increased osteoclast number/activity and decrease osteoblast number/activity (Aguirre et al., 2009; Yang et al., 2020).  

A similar trend was observed in radiation studies of 2-10 Gy X-rays, finding dose-dependent increases in empty lacunae, indicating enhanced osteocyte apoptosis under radiation exposure. The increased apoptosis of osteocytes was accompanied by significant dose-dependent increases in measures of osteoclastogenesis and decreased measures of osteoblastogenesis (Chandra et al., 2014; Wright et al., 2015). 

Many studies also examine the dose-concordance relationship between apoptosis of osteoblasts/osteoclasts and altered bone cell homeostasis under microgravity and radiation exposure. Evidence from microgravity exposures, although limited, also support the relationship. Studies show profound increases in osteoblast apoptosis in vitro, as examined by various measures, including Annexin V with FITC/PI and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stain, as well as significant increases in cleaved caspases, in-situ nick-end labeling (ISEL) or the ratio of B-cell lymphoma (Bcl)-2 to Bcl-2 associated X protein (Bax). Following microgravity, an increase in cell death in addition to an increase in osteoclast number (Aguirre et al., 2006) or TRAP-positive cells (Wu et al., 2020) and a decrease in ALP activity, a marker of bone deposition, as well as increases in measures of osteoclast bone resorption were observed (Yang et al., 2020). Data on gamma and X-ray radiation-induced osteoblast apoptosis is plentiful, with most studies examining the effects of high doses of ionizing radiation (> 2 Gy). Murine models exposed to high-dose X-ray radiation have shown increased osteoblast apoptosis under 8-12 Gy with accompanying decreased osteoblast and increased osteoclast activity (Chandra et al., 2017; Chandra et al., 2014; Liu et al., 2018). Relatively lower dose studies (0.25-4 Gy) have found significant increases in osteoblast apoptosis resulting in a decrease in ALP activity (Huang et al., 2019; Li et al., 2020; Li et al., 2015). 

One study of osteoclast apoptosis under radiation exposure has also revealed interesting results, observing significantly increased apoptosis of osteoclasts, but with enhanced osteoclast activity and bone resorption (Huang et al., 2018). It is proposed that osteoclast apoptosis results in the recruitment macrophages that release inflammatory molecules that directly activate osteoclasts and induce RANK-L expression, ultimately increasing the overall pool of osteoclasts in bone (Huang et al., 2018). 

 A study performed in osteoblasts observed significant increases in autophagy induction under ionizing radiation exposure, with decreased osteoblast activity (Li et al., 2020). 

Time Concordance 

A moderate amount of evidence exists in the current literature suggesting a time concordance relationship between apoptosis and altered bone cell homeostasis. Increases in osteoblast and osteocyte apoptosis has been observed as early as 24-72 hours post-irradiation, and as early as day 3 of microgravity exposure. The resulting effects on bone cell homeostasis under microgravity exposure have been observed by days 3-7, and under radiation exposure as early as 3 days post- exposure, indicating a slight delay in the loss of homeostasis after onset of apoptosis (Aguirre et al., 2006; Li et al., 2020; Wright et al., 2015; Yang et al., 2020). 

Essentiality 

Studies examining the effects of various countermeasures to apoptosis and autophagy of osteoblasts, osteoclasts, and osteocytes suggest a strong relationship between the occurrence of cell death and altered bone cell homeostasis. 1-34 amino-terminal fragment of parathyroid hormone (PTH)1-34 is used to treat osteoporosis by stimulating both osteoblast and osteoclast activity, but with greater stimulation of osteoblasts; it can increase bone deposition by suppressing apoptosis of mature osteoblasts. In a study of the effects of PTH1-34 treatment in mouse tibial bones exposed to 8 Gy X-ray radiation, PTH1-34 was found to fully reverse the effect of radiation on both osteoblast and osteocyte cell death and enhance overall osteoblast number under radiation exposure to vehicle-treated unirradiated controls (Chandra et al., 2014).  

α-2-macroglobulin (α2M) is a macromolecular glycoprotein found in plasma that possesses a wide range of biological functions, including radioprotective and anti- inflammatory effects. Treatment of 12 Gy X-ray irradiated hBMMSCs (osteoblast precursors), with 0.25-0.5 mg/mL of α2M was found to dose-dependently decrease cell apoptosis rate of hBMMSCs, as well as dose-dependently increased ALP activity, indicating increased induction of osteoblastogenesis in these cells, and bone deposition, as demonstrated by Alizarin red staining for calcium nodule formation (Liu et al., 2018). Another radioprotective compound known to promote healing in bone fractures is Amifostine (AMI), which protects cells from radiation-induced DNA damage by preventing interaction with reactive oxygen species. In vitro research with bone marrow-derived mesenchymal stem cells (bmMSCs) found that treatment with AMI fully reversed apoptosis induction under 2 Gy gamma radiation, as measured by Annexin V FITC/PI double staining, and ultimately restored ALP activity and calcium deposition by osteoblasts to control levels (Huang et al., 2019). 

Glucocorticoids (GCs) are known to induce devastating effects on bone mass and density by decreasing bone remodeling; the mechanism by which this occurs is through suppression of osteoblast differentiation and induction of osteoblast apoptosis. In a study examining transgenic mice, blocking GC signaling of hindlimb unloaded mice was found to fully reverse the effect of microgravity on osteoblast and osteocyte apoptosis, as well as decreasing the production of RANK-L by osteocytes. GC signaling blockade was also found to fully protect the decrease in osteoblast number observed in unloading and restore markers of osteoblast activity, as well as diminish markers of osteoclastogenesis and osteoclast number (Yang et al., 2020). 

MicroRNAs (miRNA) are a well-known tool for epigenetically modifying gene expression; many studies have shown that miRNAs may be implicated in bone cell differentiation and suppression of disuse osteopenia through various mechanisms. MiR-655-3p is a miRNA that has been proposed to prevent the induction of osteopenia in simulated microgravity. Inhibition of miR-655-3p was found to profoundly enhance osteoblast apoptosis and decrease ALP activity; microgravity- exposed cells treated with miR-655-3p were fully protected against microgravity-induced apoptosis, and had ALP activity fully restored, indicating microgravity- induced apoptosis of osteoblasts may play a role in decreased bone deposition (Wang et al., 2020b). 

One study found that inhibition of autophagy after microgravity reduces osteoclast activity. Both 4-acetylantroquinonol B (4-AAQB) and 3-methyladenine (3-MA) can inhibit autophagy induction. Treatment of osteoclasts with these autophagy inhibitors results in reduced osteoclast activity (Wu et al., 2020). 

Treatment of irradiated osteoblasts with doxycycline, an antibiotic compound that inhibits autophagy, was found to fully reverse the increased expression of autophagy proteins ATG5, Beclin-1, and LC3-II/LC3-I, while also substantially increasing ALP activity under 0.25-4 Gy radiation (Li et al., 2020). Similarly, treatment with α-2-macroglobulin, a glycoprotein with diverse cellular functions, was found to reverse radiation-induced autophagy induction and increase ALP activity, restoring them to near-control levels (Liu et al., 2018). These results suggest autophagy induction in osteoblasts may also play a role in the suppression of bone deposition observed under radiation exposure. 

• The exact mechanism by which apoptotic osteocytes recruit osteoclasts is disputed. Some studies support the notion that apoptotic osteocytes in bone cannot be engulfed by phagocytes, due to physical restriction, and thus allow for rupture of the cell membrane; this allows for the release of a variety of osteoclast stimulatory factors that directly enhance bone resorption (Jilka, Noble, and Weinstein, 2013; Komori et al., 2013). Other studies, however, propose that dying osteocytes signal to viable osteocytes in their vicinity to release osteoclast stimulatory molecules, which then enhance osteoclast activity (O’Brien, Nakashima, and Takayanagi, 2013; Plotkin, 2014). Further research in this area may aid in elucidating the mechanisms of osteoclast recruitment directed to apoptotic osteocytes. 

Dose/Incidence Concordance- Apoptosis  

Dose/Incidence concordance- Autophagy 

Time Concordance 

Not Identified