• apoptosis;
  • osteoblasts;
  • osteocytes;
  • bone formation


  1. Top of page
  2. Abstract
  9. Acknowledgements

Since the initial demonstration of the phenomenon in murine and human bone sections ∼10 yr ago, appreciation of the biologic significance of osteoblast apoptosis has contributed greatly not only to understanding the regulation of osteoblast number during physiologic bone remodeling, but also the pathogenesis of metabolic bone diseases and the pharmacology of some of the drugs used for their treatment. It is now appreciated that all major regulators of bone metabolism including bone morphogenetic proteins (BMPs), Wnts, other growth factors and cytokines, integrins, estrogens, androgens, glucocorticoids, PTH and PTH-related protein (PTHrP), immobilization, and the oxidative stress associated with aging contribute to the regulation of osteoblast and osteocyte life span by modulating apoptosis. Moreover, osteocyte apoptosis has emerged as an important regulator of remodeling on the bone surface and a critical determinant of bone strength, independently of bone mass. The detection of apoptotic osteoblasts in bone sections remains challenging because apoptosis represents only a tiny fraction of the life span of osteoblasts, not unlike a 6-mo -long terminal illness in the life of a 75-yr -old human. Importantly, the phenomenon is 50 times less common in human bone biopsies because human osteoblasts live longer and are fewer in number. Be that as it may, well-controlled assays of apoptosis can yield accurate and reproducible estimates of the prevalence of the event, particularly in rodents where there is an abundance of osteoblasts for inspection. In this perspective, we focus on the biological significance of the phenomenon for understanding basic bone biology and the pathogenesis and treatment of metabolic bone diseases and discuss limitations of existing techniques for quantifying osteoblast apoptosis in human biopsies and their methodologic pitfalls.


  1. Top of page
  2. Abstract
  9. Acknowledgements

Apoptosis is the default fate of all nucleated cells, and cell survival is maintained by several defense mechanisms designed to constantly combat external or internal proapoptotic insults. For regenerating tissues such as bone, apoptosis is as critical as the birth of new cells. Indeed, as in the case of other nucleated cells in the body, it is well documented that the number of osteoblasts, the highly specialized cells that produce and mineralize matrix, is determined by the balance between their birth from mesenchymal progenitors and their death by apoptosis.(1) During the process of regeneration, osteoblasts assemble at the bottom of resorption lacunae created by osteoclasts and begin to fill the cavities with new bone. As they move away from the cement line, they gradually flatten. Eventually, some osteoblasts become the lining cells that cover quiescent surfaces and some become osteocytes, but 60–80% of the osteoblasts that originally assembled at the resorption pit cannot be accounted for by either of these two fates.(2) Evidence produced during the last 10 yr has established that the missing osteoblasts die by apoptosis.(3)

Since osteoblast apoptosis was first shown in sections of murine and human remodeling bone almost 10 yr ago,(4) it has become increasingly evident that delay or hastening of the event plays a major role for skeletal homeostasis. Despite such evidence, some observers in our field remain skeptical about the importance of osteoblast apoptosis for bone metabolism or downplay its importance. In this perspective, we will discuss the significance of osteoblast apoptosis for understanding the pathogenesis and treatment of metabolic bone diseases.


  1. Top of page
  2. Abstract
  9. Acknowledgements

The key features of apoptotic cells are loss of volume, collapse of the cytoskeleton, nuclear fragmentation and DNA degradation, and detachment from the extracellular matrix. In addition, apoptotic cells display signals on their surface that are recognized by phagocytes for ingestion. All these changes occur without the release of intracellular contents that would otherwise provoke an inflammatory response. The entire process is highly regulated and can be triggered by extrinsic signals in which death receptors initiate the apoptosis program on binding of proapoptotic factors like Fas ligand or by intrinsic signals that ultimately disrupt the integrity of mitochondria and thereby initiate the death program.(5) The latter represents the most frequent apoptotic mechanism in vertebrates.

The most common cues for the initiation of apoptosis are genotoxic agents, oxidative stress, changes in energy metabolism, loss of kinase-mediated survival pathways activated by integrins, locally produced growth factors, cytokines, and systemic hormones. In the mitochondria pathway, the decision to live or die is determined at the level of the outer mitochondrial membrane. The integrity of this membrane is controlled by the Bcl-2 family of proteins, which are made up of pro-apoptotic and anti-apoptotic members.(6) When death signals overwhelm survival signals, the actions of anti-apoptotic Bcl-2 proteins are abrogated. This results in the permeabilization of the outer mitochondrial membrane and the release of proteins from the intermitochondrial membrane space into the cytoplasm. Most important among such proteins are cytochrome c, Omi, and Diablo. Death signals also activate p66Shc, a protein that interferes with the electron transport machinery of the inner mitochondrial membrane and thereby generates reactive oxygen species (ROS) that contribute to permeabilization of the outer mitochondrial membrane.(7)

Release of cytochrome c into the cytoplasm activates caspase-9, a protease that normally exists as a latent proenzyme.(8) Once activated, caspase-9 proceeds to activate other caspases, notably caspase-3 and caspase-7, which eventually dismantle the internal components of the cell. Omi and Diablo contribute to the process by blocking the activity of proteins like x-chromosome–linked inhibitor of apoptosis protein, which normally prevent caspase activation.

The entire process of apoptosis, from the initial insult to the complete disappearance of the cell, lasts anywhere between 2–3 h and 1 or 2 days.(9,10) Apoptotic cells vanish without a trace as they are cleared by phagocytes in a manner so efficient that they are difficult to find in typical tissue sections. Nevertheless, a few apoptotic cells can signify extensive cell loss, analogous to the increase in cell proliferation that is heralded by finding a few mitotic figures in a tumor.(11)


  1. Top of page
  2. Abstract
  9. Acknowledgements

The presence of degraded DNA is a convenient and relatively specific marker of cells undergoing apoptosis. Techniques for visualizing cells with degraded DNA are known by a number of acronyms that are often used interchangeably, the most common being TUNEL for terminal deoxynucleotidyl transferase mediated dUTP nick-end-labeling and ISEL for in situ end-labeling. These methods involve attachment of a labeled nucleotide to hydroxyl groups at the 3′ end of DNA using either terminal deoxynucleotidyl transferase or the Klenow fragment of DNA polymerase I. Use of the Klenow enzyme detects cells with fewer DNA strand breaks, and labeling with this enzyme is less susceptible to artifact because it lacks exonuclease activity.(12) As in any quantitative histologic procedure, detection of degraded DNA in apoptotic cells requires standardization and the use of appropriate negative and positive controls during each staining run. In Table 1, we list guidelines that we follow for obtaining reproducible estimates of apoptotic osteoblasts, based on the experience of others as well as our own over the last 10 yr, using the TUNEL procedure with different murine models.

Table Table 1.. Guidelines for the Detection of Apoptotic Osteoblasts
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In vertebral cancellous bone of adult mice, 0.5–1.0% of osteoblasts exhibit DNA strand breaks when analyzed with the terminal deoxynucleotidyl transferase enzyme.(4,13,14) Using the more sensitive Klenow enzyme, the prevalence of apoptotic osteoblasts increases to as much as 5–10%.(15,16) Longitudinal sections of lumbar vertebrae (L1–L4) of adult mice are particularly advantageous samples for quantifying osteoblast apoptosis because they typically contain 800–1200 of osteoblasts for inspection. This situation allows statistically meaningful estimates of the prevalence of apoptosis.

How can a phenomenon of such low prevalence be important? The phase of apoptosis that can be detected by TUNEL staining represents only a tiny fraction of the cell's life span, analogous to the brevity of a 6-mo-long terminal illness compared with the entire life of a 75-yr-old human. Tidball and Albrecht(17) have estimated that an entire tissue could die of apoptosis in 20 days if only 0.4% of the nuclei in the tissue were detectably apoptotic (using the terminal deoxynucleotidyl transferase enzyme) at any given time. This calculation is based on the assumption that there is a 2-h period of detectability of the phenomenon, that apoptosis occurs at a constant rate, and that no mitosis occurred during this period. Based on the fact that in the snapshot of a biopsy the fraction of cells exhibiting apoptotic features is equal to the corresponding fraction of time spent in apoptosis, one can calculate the percentage of cells that undergo apoptosis from the following equation:

  • equation image

Osteoblast life span can be determined by dividing the wall width (the histologic measure of the amount of bone made by a team of osteoblasts) by the mineral apposition rate. For osteoblasts in cancellous bone of normal adult mice, this is ∼12 days.(14) It is unknown how long the DNA fragmentation phase of apoptosis lasts in dying osteoblasts, but in liver cells, the duration of this particular phase is 2–3 h when visualized with the terminal deoxynucleotidyl transferase method.(9) There is no reason to expect that this phenomenon would substantially vary among cell types. Therefore, if the prevalence of murine osteoblast apoptosis is 0.6%, it can be calculated that 60–90% of osteoblasts die by apoptosis; the rest become osteocytes or lining cells. This estimate is very similar to the estimate of Parfitt(2) for osteoblasts that die by apoptosis in human bone.

Despite this sensible and comforting biological parity between humans and mice, the prevalence of osteoblast apoptosis in human bone is much lower compared with mice. This difference results from two reasons: human osteoblasts live longer and are fewer in number compared with their murine counterparts because bone turnover in humans is lower. Specifically, whereas the average osteoblast life span is ∼12 days in normal adult mice,(14) the average life span of normal human osteoblasts is ∼150 days(18)—a 12.5-fold difference. Therefore, a 0.6% prevalence of osteoblast apoptosis in mice corresponds to a prevalence of 0.05% in humans. Furthermore, whereas the average bone formation rate in Swiss-Webster mice is ∼0.150 μm2/μm/d,(14) the corresponding value in healthy humans is ∼0.038 μm2/μm/d(19)—a 4-fold difference. There is no reason to suspect that the duration of the morphologic features of apoptosis is different in the two species. Likewise, in both species, the rate of bone formation is inexorably proportional to the number of osteoblasts.(20) That is to say, the higher the number of osteoblasts present in a defined area of bone, the greater the chance to detect an apoptotic one. If the relationship between osteoblast number and bone formation rate is similar in the two species (once again, there is no reason to suspect that it is different), the 12-fold difference in life span between the two species needs to be multiplied by the 4-fold difference in bone formation rate. This simple calculation makes the chance of seeing an apoptotic osteoblast in a section of human bone ∼50 times lower than in a section of murine bone.

The total number of osteoblasts present in a longitudinal section obtained from a transiliac biopsy from humans is only 20–50 cells(21) compared with the 800–1200 cells present in a single longitudinal section of four murine vertebrae, typically used in our laboratory for the analysis of osteoblast apoptosis. If one were to estimate the prevalence of apoptosis—a phenomenon that is predicted to occur in 0.01–0.1% of the entire osteoblast population—relying on the inspection of 20–50 cells present in a section from a human biopsy, one will fail to find a single apoptotic cell most of the time. In other words, to achieve comparable statistical power for estimating the prevalence of osteoblast apoptosis in a human bone section as in a murine section, one would have to prepare 100 sections and stain them with the more sensitive Klenow enzyme. Such sections are usually 5–7 μm thick (the optimal usable thickness for staining and microscopy) and must be nonadjacent because osteoblast height is 12–20 μm. Alas, this is practically impossible to achieve with a normal size human transiliac biopsy of 7 mm diameter. Not surprisingly, despite our experience with murine bone, we have been unable to detect a single apoptotic osteoblast in transiliac biopsies taken from 12 normal subjects using the less sensitive TUNEL enzyme.(14) The more sensitive Klenow enzyme was used to search for apoptotic osteoblasts in biopsies from 23 placebo-treated postmenopausal women in another study,(21) but none were found (RS Weinstein, unpublished observations, 2005).

There are only two scientifically valid ways of estimating rare events like apoptosis. The first is enumeration of a vast number of cells in which the sparse phenomenon may happen. The second is reliance on a simulation model based on a predetermined estimate of the probability of the rare event happening, which is the only practical option for human biopsies, perhaps with the exception of conditions, such as glucocorticoid excess, in which the prevalence of the phenomenon increases dramatically. However, the sine qua non conditions under which such a model can be relied on is that the apoptotic events must be randomly spaced and independent of each other. Unfortunately, neither condition is certain. In fact, osteoblast apoptosis evidently occurs in clusters (Fig. 1A). As elegantly explained in the case of the detection of another rare occurrence in bone samples—namely microcracks—frequency distributions are typically skewed, with a few specimens exhibiting many events but most having only a few or none.(22) Therefore, if only a few apoptotic osteoblasts are identified in a specimen, it is a warning sign that the sample size is not large enough to estimate the prevalence of the phenomenon in the entire skeleton of an experimental group. As we explain above, an a priori estimate of the prevalence of apoptosis in advance of an experiment, together with the appropriate positive and negative controls, must be used to derive a reliable estimate of the required sample size.

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Figure Figure 1. Apoptotic osteoblasts in human and murine cancellous bone. (A) Human bone taken from a patient treated with long-term prednisone. Cuboidal apoptotic osteoblasts with dark brown condensed nuclei and a prominent perinuclear Golgi apparatus are seen adjacent to normal osteoblasts on the bone perimeter (black solid arrows). An apoptotic osteocyte is noted in the lower right corner (asterisk). The sausage-shaped cells with brown nuclei that are not on the bone perimeter cannot be positively identified as apoptotic osteoblasts and should not be counted as part of the osteoblast team (yellow block arrows). Undecalcified section stained by ISEL with the Klenow enzyme and methyl green viewed with oil immersion and Nomarski differential interference contrast microscopy (original magnification, ×1000). (B) Murine bone from an ovariectomized adult animal. Undecalcified section stained by ISEL with the Klenow enzyme and methyl green viewed with Nomarski differential interference contrast microscopy (original magnification, ×630).

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  1. Top of page
  2. Abstract
  9. Acknowledgements

Substantial evidence indicates that osteoblast apoptosis is an actively controlled process that occurs when pro-apoptotic signals exceed anti-apoptotic signals. As we will highlight below, most, if not all, major regulators of skeletal homeostasis influence the apoptosis of osteoblasts and osteocytes. Importantly, genetic factors that determine bone mass also control osteoblast apoptosis as evidenced by the inverse relationship between bone formation rate and osteoblast apoptosis in high bone mass C3H/HeJ mice compared with low bone mass C57BL/6 mice.(23) Osteoblast apoptosis is highly regulated during fracture repair and during the closing of skeletal defects in rodent models.(24–26) Interestingly, in mice with partial deficiency in hypoxia-inducible factor 1α (a transcription factor involved in the regulation of anaerobic metabolism and angiogenesis), the callus of experimentally induced femoral fractures is larger, and osteoblast apoptosis is decreased.(27) Thus, regulation of osteoblast apoptosis during fracture healing seems to play a role in modulating the pace of bone regeneration.

It is also important to note that apoptosis can occur throughout the entire life span of osteoblasts, beginning from the early stages of differentiation and continuing throughout all stages of the working life of this cell. Evidence for this is provided by the observation of apoptotic mesenchymal progenitors near the primary spongiosa of developing chick and rabbit long bones,(28) the osteogenic front of developing murine calvarial bone,(29) at sites of fracture healing,(24,25) and near the newly forming bone that develops during distraction osteogenesis—a process in which long bones are cut and gradually stretched, resulting in de novo bone formation within the expanding gap.(30) Cells in other regenerating tissues exhibit similar behavior. For example, intestinal epithelial cells exhibit DNA fragmentation 1–2 days before their appearance at the tip of the microvillus where they all die by apoptosis,(10) and some chondrocytes undergo apoptosis during the proliferative stage, well before they become frankly apoptotic hypertrophic chondrocytes.(31)

Growth factors, cytokines, and integrins

Bone morphogenetic proteins (BMPs) induce apoptosis of mesenchymal osteoblast progenitors in interdigital tissues during the development of hands and feet.(32) Interestingly, BMP-2 induces apoptosis of cultured osteoblastic cells in a manner that is independent of its pro-differentiating effect.(33,34)

Wnt signaling has a profound effect on osteoblasts as exemplified by the high bone mass phenotype of mice and humans with activating mutations of low-density lipoprotein receptor-related protein 5 (LRP5), which acts as a co-receptor with Frizzled for Wnt protein ligands. Besides their well-established role in the proliferation and differentiation of osteoblast progenitors, Wnts are also involved in the control of osteoblast apoptosis. Indeed, Wnt signaling directly activates anti-apoptosis pathways in osteoblastic cell models.(35) More important, mice with the G171V activating mutation of LRP5 exhibit increased bone formation that is associated with decreased osteoblast and osteocyte apoptosis.(36) The expression of secreted frizzled related protein-1 (SFRP-1), an antagonist of Wnt signaling, increases with increasing osteoblast maturation,(37) and deletion of SRFP-1 causes an increase in bone formation associated with decreased osteoblast apoptosis.(38)

TGF-β, IGF-I, fibroblast growth factor-2 (FGF-2), and interleukin-6 (IL-6) type cytokines inhibit apoptosis of cultured osteoblastic cells.(4,39–41) Conversely, mice lacking Smad-3, which mediates TGFβ signaling, exhibit decreased bone mass associated with increased osteoblast apoptosis.(42) Fas ligand, IL-1, and TNF, on the other hand, stimulate apoptosis of osteoblastic cells.(4,43)

Interaction of cultured osteoblastic cells with the extracellular matrix generates anti-apoptosis signaling through integrins.(44,45) Moreover, mice expressing a matrix metalloproteinase (MMP)-resistant mutant of type I collagen exhibit increased osteoblast and osteocyte apoptosis. Thus, MMP-mediated release of growth factors from the extracellular matrix or exposure of cryptic integrin binding sites in the matrix must also contribute to anti-apoptotic signaling in these cells.(46)


Estrogens and androgens prevent osteoblast and osteocyte apoptosis through a mechanism of action that is mediated by the classical estrogen or androgen receptors and involves kinase-mediated survival signaling(47) and downstream transcription factors such as Elk-1, C/EBPβ, CREB, and c-jun.(48) Interestingly, 1,25-dihydroxyvitamin D3 also inhibits apoptosis of osteoblastic cells through activation of similar kinase-mediated signaling pathways.(49)

Consistent with the in vitro findings, acute loss of estrogen in women and rodents causes an increase in osteocyte apoptosis,(47,50,51) and gonadectomy of male or female mice causes an increase in both osteoblast and osteocyte apoptosis.(52) In contrast, sex steroids promote osteoclast apoptosis, and sex steroid deficiency increases the life span of osteoclasts.(52–54) The opposite effects of sex steroids on the life span of osteoblasts/osteocytes and osteoclasts help to maintain a focal balance between formation and resorption. The anti-apoptotic effect of sex steroids on osteocytes may also contribute to the antifracture efficacy of sex steroids independently of their effect on BMD, but this notion has not been tested directly.

Glucocorticoid excess increases the prevalence of osteoblast and osteocyte apoptosis in murine vertebrae and human iliac bone alike.(14,55) This may explain, at least in part, the decrease in osteoblast number and bone formation rate caused by glucocorticoid excess. In this condition, osteocytes with condensed chromatin have been observed between the tetracycline labeling that demarcates sites of bone formation, indicating that they died immediately after entombment in the bone matrix.(56) The pro-apoptotic effect of glucocorticoids on osteoblast and osteocyte apoptosis is evidently direct, because overexpression in osteoblasts of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), an enzyme that inactivates glucocorticoids, prevents the steroid-induced increase in apoptosis.(57) Moreover, osteoblast number, osteoid area, and bone formation rate are significantly higher in glucocorticoid-treated 11β-HSD2 transgenic mice compared with glucocorticoid-treated wildtype controls.

Glucocorticoid-stimulated apoptosis of cultured osteoblastic and osteocytic cells is strictly dependent on the glucocorticoid receptor (GR),(16,55) and osteoblast-specific deletion of the GR prevents glucocorticoid-induced osteoblast apoptosis.(58) Strikingly, in an osteocytic cell model (MLO-Y4), the pro-apoptotic effect of glucocorticoids is preceded by cell detachment caused by interference with focal adhesion kinase (FAK)-mediated survival signaling generated by integrins. In this mechanism, Pyk2 (a member of the FAK family) becomes phosphorylated and subsequently activates pro-apoptotic JNK signaling.(59) In addition, the pro-apoptotic actions of glucocorticoids may involve suppression of the synthesis of locally produced anti-apoptotic factors including IGF-I and IL-6 type cytokines, as well as MMPs,(60) and stimulation of the proapoptotic Wnt antagonist SFRP-1.(61) Interestingly, different isoforms of the GR, when transfected into human osteoblastic cells, have quantitatively different effects on apoptosis (JA Cidlowski, personal communication, 2007). Glucocorticoids exert the exactly opposite effect on osteoclast apoptosis (i.e., glucocorticoid excess delays osteoclast apoptosis),(62) and this effect may account for the early transient increase in bone resorption in patients with exogenous or endogenous hyperglucocorticoidism.

Daily injections of PTH or PTH-related protein (PTHrP) have well-known anabolic effects on the skeleton. Studies in humans indicate that most of the increase in bone formation with such regimens occurs within preexisting basic multicellular units, and is associated with spillage of osteoblasts outside the boundary of the bone surface being actively remodeled.(63,64) Daily administration of 3–300 ng/g of PTH to mice causes a dose-dependent increase in osteoblast number, bone formation rate, circulating osteocalcin, and BMD.(15) These changes are inversely correlated with a reduction in the prevalence of osteoblast apoptosis (Fig. 2). After only two injections, the prevalence of apoptotic osteoblasts declines from 11% to 8%. After four injections, there is a further decline to 5%, and osteoblast number increases by 2- to 3-fold. Thus, increased survival is a major contributor to the increase in osteoblast number caused by intermittent PTH in cancellous bone. Intermittent PTH also stimulates osteoblast differentiation by causing withdrawal of osteoblast progenitors from the cell cycle, transiently activating Runx2, and increasing production and/or activation of locally produced growth factors.(65) Mice with a deletion of PTHrP from cells of the osteoblast lineage exhibit an increased number of apoptotic osteoblasts and decreased osteoblastogenesis.(66,67) Thus, it seems that daily injections of PTH add to the anti-apoptotic and osteoblastogenic signals of endogenous PTHrP.

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Figure Figure 2. Inverse relationship between osteoblast apoptosis and bone formation. Each point corresponds to the value of serum osteocalcin, bone formation rate, or osteoblast number determined in the serum or femur of a single animal plotted as a function of the prevalence of osteoblast apoptosis in the same animal. Linear regression lines (±95% CIs) are shown. Pearson product moment analysis was used to determine the strength of the correlation (r) and its significance level (p < 0.0001). Reprinted with permission from Bellido et al.(15)

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PTH and PTHrP inhibit apoptosis in cultures of rat, murine, and human osteoblastic cells(4,15,68) through cAMP-activated protein kinase A, inactivation of the pro-apoptotic protein Bad, and increased transcription of survival genes like Bcl-2.(15) The increased synthesis of survival genes requires cAMP response element-binding protein and Runx2. Besides PTH and PTHrP, other hormones that stimulate cAMP production in osteoblastic cells, including calcitonin and prostaglandin E, inhibit apoptosis of osteoblastic cells.(16,69)

In sharp contrast to the evidence for attenuation of osteoblast apoptosis by intermittent PTH administration in mice, in a recent study of transiliac biopsies from postmenopausal women receiving daily injections of PTH for 28 days, Lindsay et al.(70) found an increase in osteoblast apoptosis in cancellous bone, as well as a positive correlation between osteoblast apoptosis and bone formation rate. In our opinion, it is highly unlikely that a species difference can account for this incongruent result. The absolute number of apoptotic osteoblasts could increase with the stimulation of osteoblastogenesis in response to intermittent PTH as Lindsay et al. suggest, yet the prevalence of apoptosis should be unaffected or reduced when expressed per number of osteoblasts. However, osteoblast apoptosis expressed per millimeter of osteoblast surface, as was done in the study of Lindsay et al., does not permit estimation of prevalence because it ignores the variance in osteoblast size that occurs during their transition from plump cuboidal, at the beginning of the refilling of a resorption cavity, to brick-like rectangular, near the end of the process.(2) Increased detection of osteoblast apoptosis in the study of Lindsay et al. could have resulted from misidentifying as osteoblasts TUNEL+ cells that are not part of the osteoblast team on the osteoid surface of cancellous bone. As shown in the human bone section depicted in Fig. 1A, apoptotic cells in close proximity to the team of osteoblasts are sometimes encountered between the osteoblast layer and the bone marrow. By virtue of the fact that such cells are not attached to the bone surface, they cannot be bone-forming osteoblasts. In addition, nonspecific staining can be easily misinterpreted as TUNEL+ even though the cells do not exhibit morphological changes typical of apoptotic osteoblasts, unless one has a standard specimen with a known CV for osteoblast apoptosis, stained simultaneously with the test sample (positive control). In agreement with Lindsay et al., we believe that more extensive studies of the effect of PTH on osteoblast survival in humans are needed. Such studies should be ideally done under conditions in which the baseline prevalence of the phenomenon is substantially increased (glucocorticoid excess) to facilitate detection of the effect of a pharmacologic manipulation.

Aging and oxidative stress

We have recently determined that both female and male C57BL/6 mice lose bone strength and mass progressively between 4 and 31 mo of age. These changes are temporally associated with a decreased rate of remodeling as evidenced by decreased osteoblast and osteoclast numbers and decreased bone formation rate, as well as decreased wall width and increased osteoblast and osteocyte apoptosis.(71,72) These changes are also temporally linked with increased ROS levels and decreased activity of the antioxidant enzyme glutathione reductase, as well as a corresponding increase in the phosphorylation of p53 and p66Shc, two key components of a signaling cascade that is activated by ROS and influences apoptosis and life span in invertebrates and mammals.

During the last few years, FOXOs, a subclass of a large family of forkhead proteins characterized by the presence of a winged-helix DNA binding domain called Forkhead Box, have been shown to play a major role in stem cell apoptosis and longevity.(73–80) FOXOs counteract the adverse effects of ROS by upregulating free radical scavenging enzymes such as Mn superoxide dismutase and catalase, as well as DNA-damage repair genes such as Gadd45.(79,81–83) Importantly, Essers et al(74) recently showed that FOXO-mediated transcription requires binding of β-catenin, a scaffold protein that is also needed for the transcriptional activity of the TCF-family of transcription factors, which are the downstream effectors of the Wnt/β-catenin signaling pathway.(84,85) Moreover, Tothova et al.(86) have provided compelling evidence from studies of hematopoiesis in mice with conditional deletion of FOXO1, FOXO3, and FOXO4 that FOXO deficiency increases both apoptosis of hematopoietic stem cells and terminal differentiation at the expense of self-renewal. More important, these authors were able to reverse completely the changes of the FOXO-deficient hematopoietic stem cell (HSC) phenotype by the administration of the antioxidant N-acetyl cysteine, showing that physiologic oxidative stress is a critical determinant of the survival and thereby long-term regenerative potential of stem cells.

Mesenchymal stem cell osteoblast progenitors could very well be regulated by oxidative stress in a similar manner as the HSC. Indeed, we have determined that ROS antagonize the skeletal effects of Wnt/β-catenin in vitro by diverting β-catenin from TCF- to FOXO-mediated transcription.(87) Moreover, consistent with the notion that increased ROS production with age attenuates Wnt/β-catenin signaling, Gadd45—a DNA repair gene induced by FOXO—is increased, whereas Axin2 and osteoprotegerin mRNA are decreased in old compared with young C57BL/6 mice. Activation of Wnt signaling enables osteoblastogenesis, inhibits adipogenesis and osteoclastogenesis, and increases bone mass. In addition, Wnts prevent apoptosis of both uncommitted osteoblast progenitors and differentiated osteoblasts by β-catenin–dependent and –independent signaling cascades involving Src/ERK and PI3K/Akt.(35) Therefore, diversion of β-catenin from TCF to FOXO in response to oxidative stress is in our opinion an important molecular mechanism of the age-dependent decline of osteoblastogenesis, the reciprocal increase in adipogenesis in the aging bone marrow, and the increase in osteoblast and osteocyte apoptosis.


  1. Top of page
  2. Abstract
  9. Acknowledgements

Unlike osteoblasts that begin to die by apoptosis almost as soon as they are born, most osteocytes remain alive until the bone in which they reside is replaced. Nevertheless, osteocytes do die by apoptosis as evidenced by a 2–5% prevalence of the phenomenon using the sensitive Klenow enzyme labeling method.(88) In distinction to osteoblasts, osteocyte apoptosis represents cumulative death because the cellular debris cannot be removed by phagocytes. Indeed, degraded DNA can be detected in osteocyte lacunae of necrotic human bone long after the initial insult of glucocorticoid excess.(56)

Several lines of evidence indicate that survival signaling in osteocytes is initiated by mechanical forces. Studies in rodents have shown that osteocytes exhibit a U-shaped relationship between apoptosis and strain. Specifically, osteocyte apoptosis is induced by hindlimb unloading,(88) whereas apoptosis is suppressed by application of moderate physiologic levels of strain.(89) In contrast, osteocyte apoptosis is stimulated by application of strain at levels that induce microdamage within the bone matrix.(89,90) It has been speculated that such microdamage interferes with transmission of strain through the bone. If so, osteocytes in the immediate vicinity of the microdamage may experience reduced strain and thereby undergo apoptosis.

Osteocytes interact with the extracellular matrix through integrins,(91,92) as well as transverse elements that tether them to the canalicular wall.(93) Fluid movement through the canaliculi resulting from mechanical loading may induce deformation of the extracellular matrix, generate tension in the tethering elements, or directly deform the cell membrane.(93,94) These changes may be transduced into survival signals through integrin clustering and subsequent assembly of a signalsome, resulting in extracellular signal-regulated kinase–mediated survival signaling.(95)

Moreover, dying osteocytes evidently serve as beacons for initiation of targeted bone remodeling in response to physiologic or excessive strain to prevent accumulation of microdamage that would otherwise compromise bone strength.(96) Indeed, recent studies have shown that osteocyte apoptosis precedes recruitment of osteoclasts to the area where osteocytes have died after unloading(88) or application of excessive strain.(90) Targeted remodeling with net bone loss or gain in response to unloading or excessive loading, respectively, may adjust bone mass to that needed to bear the perceived strain.

Glucocorticoid-induced osteoporosis in humans is often complicated by the in situ death of portions of bone, associated with abundant apoptotic osteocytes juxtaposed to the subchondral fracture crescent—a ribbon-like zone of collapsed trabeculae.(56) In this situation, osteocyte apoptosis represents a cumulative and irreparable defect that would disrupt the mechanosensory osteocyte–canalicular network and prevent repair of microfractures. This situation would promote collapse of the femoral head and explain the correlation between total steroid dose and the incidence of avascular necrosis of bone,(97) as well as the occurrence of osteonecrosis after cessation of steroid therapy. Studies from our group have shown that blockade of glucocorticoid action on osteoblasts and osteocytes by overexpression of 11β-HSD2 preserved osteocyte viability and bone strength despite loss of bone mass.(57) This observation suggests that osteocytes contribute to bone strength independently of bone mass. Although the mechanism(s) involved have not been established, disruption of the lacunocanalicular system that occurs with osteocyte apoptosis can lead to changes in bone material properties.(98)


  1. Top of page
  2. Abstract
  9. Acknowledgements

Small changes in the prevalence of osteoblast apoptosis in a bone section indicate extensive changes in the absolute number of these cells in the live tissue. The importance of this biologic event is well highlighted by evidence that hormones, locally produced factors like IL-6 type cytokines, IGF-I, Wnts and their antagonists, PTHrP, mechanical forces, and oxidative stress—all the major regulators of bone homeostasis—modulate osteoblast and osteocyte apoptosis. Nonetheless, accurate estimation of apoptotic osteoblasts requires careful attention to methodologic details and a bone sample containing adequate numbers of osteoblasts. Evidence obtained mostly in mice strongly indicates that bone loss caused by sex steroid deficiency, glucocorticoid excess, or aging is caused in part by increased osteoblast apoptosis. Moreover, the increase osteoblast number and bone formation rate that occurs in response to intermittent PTH may be caused in part by a reduction in osteoblast apoptosis. Confirmation of the latter finding in humans will require optimization of the existing methodology. Osteocytes also undergo apoptosis, and this occurrence decreases the mechanical competence of the bone independently of bone mass. Unlike the situation with osteoblasts, the prevalence of apoptotic osteocytes in a bone section reflects cumulative cell death, resulting from the fact that they cannot undergo phagocytosis by macrophages because of their anatomic isolation. Osteocyte apoptosis caused by reduced mechanical strain may recruit osteoclasts to the vicinity, thereby initiating the repair of the damaged bone.


  1. Top of page
  2. Abstract
  9. Acknowledgements

The authors acknowledge the support of the NIH (P01 AG13918), the Department of Veterans Affairs (Merit Review grants to RLJ, RSW, and SCM), and Tobacco Settlement funds provided by the UAMS College of Medicine for their research.


  1. Top of page
  2. Abstract
  9. Acknowledgements
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