Regulatory Mechanisms of RANKL Presentation to Osteoclast Precursors

It is important to understand the molecular mechanisms regulating osteoclast formation, as excess activation of osteoclasts is associated with various osteopenic disorders. Receptor activator of nuclear factor kappa B (RANKL) is a central player in osteoclastogenesis. Recent findings suggest that osteocytes are the major supplier of RANKL to osteoclast precursors. It has also been suggested that osteocyte cell death upregulates the RANKL/osteoprotegerin (OPG) ratio in viable osteocytes adjacent to apoptotic osteocytes in areas of bone microdamage, thus, contributing to localized osteoclast formation. Indeed, viable osteocytes can provide RANKL through direct interactions with osteoclast precursors at osteocyte dendritic processes. In addition, OPG tightly regulates RANKL cell surface presentation in osteocytes, which contributes to the inhibition of RANKL signaling, as well as the decoy receptor function of OPG. By contrast, the physiological role of RANKL in osteoblasts is yet to be clarified, although similar mechanisms of regulation are observed in both osteocytes and osteoblasts.     

Cell autonomous requirement of connexin 43 for osteocyte survival: consequences for endocortical resorption and periosteal bone formation

Connexin 43 (Cx43) mediates osteocyte communication with other cells and with the extracellular milieu and regulates osteoblastic cell signaling and gene expression. We now report that mice lacking Cx43 in osteoblasts/osteocytes or only in osteocytes (Cx43(ΔOt) mice) exhibit increased osteocyte apoptosis, endocortical resorption, and periosteal bone formation, resulting in higher marrow cavity and total tissue areas measured at the femoral mid-diaphysis. Blockade of resorption reversed the increased marrow cavity but not total tissue area, demonstrating that endocortical resorption and periosteal apposition are independently regulated. Anatomical mapping of apoptotic osteocytes, osteocytic protein expression, and resorption and formation suggests that Cx43 controls osteoclast and osteoblast activity by regulating osteoprotegerin and sclerostin levels, respectively, in osteocytes located in specific areas of the cortex. Whereas empty lacunae and living osteocytes lacking osteoprotegerin were distributed throughout cortical bone in Cx43(ΔOt) mice, apoptotic osteocytes were preferentially located in areas containing osteoclasts, suggesting that osteoclast recruitment requires active signaling from dying osteocytes. Furthermore, Cx43 deletion in cultured osteocytic cells resulted in increased apoptosis and decreased osteoprotegerin expression. Thus, Cx43 is essential in a cell-autonomous fashion in vivo and in vitro for osteocyte survival and for controlling the expression of osteocytic genes that affect osteoclast and osteoblast function.     

Osteocyte Apoptosis and Osteoclast Presence in Chicken Radii 0–4 Days Following Osteotomy

Osteocyte apoptosis caused by load-induced microdamage is followed by osteoclastic bone remodeling, and a causal link between apoptosis and repair has been suggested. The objectives of the present study were to use a chick model to examine the incidence of osteocyte apoptosis and the presence of osteoclasts during the first 96 hours following an osteotomy, prior to extensive callus mineralization. Osteotomies were performed on the right radii of 24 chicks at 23–24 days of age. The left radii served as controls. Radii were collected and processed at six time points following surgery (0, 12, 24, 48, 72, and 96 hours). Decalcified bone tissue sections were stained either for apoptosis using a modified TUNEL procedure or for tartrate-resistant acid phosphatase to identify osteoclasts in the intracortical and periosteal envelopes. The percentage of apoptotic osteocytes, as well as osteoclast counts (n/mm or n/mm²) were quantified in four regions (0–1, 1–2, 2–4, and 4–8 mm from the site of the osteotomy; regions 1–4, respectively) in the osteotomized radii and in the same measured areas in the control radii. Data for osteocyte apoptosis and osteoclasts in the control limb were subtracted from the osteotomized limb data to identify differences due to surgical influence. The incidence of osteocyte apoptosis was significantly higher at 12, 24, 48, and 72 hours versus 0 hours following osteotomy, and the response was highest in region 1; however, there was no interaction between time and region. Intracortical osteoclast counts (n/mm²) were elevated after 48 hours, and the response was similar in all regions. The data demonstrate that osteocyte apoptosis occurs within 24 hours in response to an osteotomy and temporally precedes an increase in osteoclast presence. Hence, osteocyte apoptosis may play a role in signaling during the bone healing process.     

Activation of resorption in fatigue-loaded bone involves both apoptosis and active pro-osteoclastogenic signaling by distinct osteocyte populations

Osteocyte apoptosis is required to initiate osteoclastic bone resorption following fatigue-induced microdamage in vivo; however, it is unclear whether apoptotic osteocytes also produce the signals that induce osteoclast differentiation. We determined the spatial and temporal patterns of osteocyte apoptosis and expression of pro-osteoclastogenic signaling molecules in vivo. Ulnae from female Sprague-Dawley rats (16-18weeks old) were cyclically loaded to a single fatigue level, and tissues were analyzed 3 and 7days later (prior to the first appearance of osteoclasts). Expression of genes associated with osteoclastogenesis (RANKL, OPG, VEGF) and apoptosis (caspase-3) were assessed by qPCR using RNA isolated from 6mm segments of ulnar mid-diaphysis, with confirmation and spatial localization of gene expression performed by immunohistochemistry. A novel double staining immunohistochemistry method permitted simultaneous localization of apoptotic osteocytes and osteocytes expressing pro-osteoclastogenic signals relative to microdamage sites. Osteocyte staining for caspase-3 and osteoclast regulatory signals exhibited different spatial distributions, with apoptotic (caspase 3-positive) cells highest in the damage region and declining to control levels within several hundred microns of the microdamage focus. Cells expressing RANKL or VEGF peaked between 100 and 300μm from the damage site, then returned to control levels beyond this distance. Conversely, osteocytes in non-fatigued control bones expressed OPG. However, OPG staining was reduced markedly in osteocytes immediately surrounding microdamage. These results demonstrate that while osteocyte apoptosis triggers the bone remodeling response to microdamage, the neighboring non-apoptotic osteocytes are the major source of pro-osteoclastogenic signals. Moreover, both the apoptotic and osteoclast-signaling osteocyte populations are localized in a spatially and temporally restricted pattern consistent with the targeted nature of this remodeling response.     

Osteocytes subjected to fluid flow inhibit osteoclast formation and bone resorption

Bone has the capacity to alter its mass and structure to its mechanical environment. Osteocytes are the predominant bone cells and it is generally accepted that the osteocytes are the professional mechanosensors of bone. A strain-derived fluid flow through the lacuno-canalicular porosity seems to mechanically activate them, resulting in the production of signalling molecules such as nitric oxide (NO). We hypothesize that mechanically stimulated osteocytes modulate osteoclast formation and activity via soluble factors, thus affecting bone resorption. Osteocytes, osteoblasts, and periosteal fibroblasts were isolated from fetal chicken calvariae via enzymatic digestion. The periosteal fibroblasts were obtained from the periostea. Osteocytes were separated from osteoblasts by immunomagnetic separation. Cells were mechanically stimulated for 1 h with pulsating fluid flow (PFF, 0.70 +/- 0.30 Pa) at 5 Hz, or kept under static conditions. Conditioned medium was collected after 60 min. The effect of conditioned medium on osteoclastogenesis was tested on mouse bone marrow cells in the presence of macrophage colony stimulating factor and receptor activator of NF-kappaB ligand. After 6 days of culture, osteoclast formation and bone resorption was determined. Osteocytes subjected to 1 h pulsating fluid flow produced conditioned medium that inhibited the formation of osteoclasts. For osteoblast PFF-conditioned medium, such effect was, to a lesser extent, also observed, but not for periosteal fibroblast PFF-conditioned medium. Furthermore, PFF-treated osteocytes, but not osteoblast or periosteal fibroblast, produced conditioned medium that resulted in a decreased bone resorption. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester attenuated the inhibitory effects of osteocyte PFF-conditioned medium on osteoclast formation and resorption. We conclude that osteocytes subjected to PFF inhibit osteoclast formation and resorption via soluble factors, and the release of these factors was at least partially dependent on activation of an NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast regarding to the production of soluble factors affecting osteoclast formation and bone resorption.     

Dissociation of the pro-apoptotic effects of bisphosphonates on osteoclasts from their anti-apoptotic effects on osteoblasts/osteocytes with novel analogs

Bisphosphonates induce osteoclast apoptosis, thereby decreasing bone resorption and reducing the rate of bone remodeling. Earlier work from our group and others has demonstrated that, additionally, bisphosphonates prevent osteoblast and osteocyte apoptosis in vivo and in vitro, raising the possibility that perhaps part of their anti-fracture efficacy may result from preserving the integrity of the osteocyte network and prolonging the working time of bone forming cells. Whereas induction of osteoclast apoptosis results from inhibition of the mevalonate pathway or from conversion to toxic ATP analogs, prevention of osteoblastic cell apoptosis is mediated by connexin43 hemichannel opening and activation of the extracellular signal-regulated kinases (ERKs). We examined here the ability of several bisphosphonates, including novel analogs, to exert these two effects. All 16 bisphosphonates studied inhibited etoposide-induced apoptosis of MLO-Y4 osteocytic cells and osteoblastic cells derived from calvaria, with EC50 between 10(-12) and 10(-10) M. On the other hand, only 10 analogs induced apoptosis of RAW-264.7-cell-derived osteoclasts. Each of the 6 bisphosphonates that lack pro-apoptotic activity in osteoclasts but retain anti-apoptotic activity in osteoblasts and osteocytes has a structural-related analog that is active in both cell types. These findings indicate that the structural prerequisites for the anti-apoptotic effect of bisphosphonates on cells of the osteoblastic lineage are less stringent than the ones required to induce osteoclast apoptosis and confirm that bisphosphonates act on the two cell types by distinct mechanisms. Preservation of osteoblast and osteocyte viability without inducing osteoclast apoptosis by these bisphosphonates analogs opens new possibilities for the treatment of bone fragility in conditions in which a decrease in bone remodeling is not desirable.     

Demonstration of osteocytic perilacunar/canalicular remodeling in mice during lactation

Osteoclasts are thought to be solely responsible for the removal of bone matrix. However, we show here that osteocytes can also remove bone matrix by reversibly remodeling their perilacunar/canalicular matrix during the reproductive cycle. In contrast, no osteocytic remodeling was observed with experimental unloading despite similar degrees of bone loss. Gene array analysis of osteocytes from lactating animals revealed an elevation of genes known to be utilized by osteoclasts to remove bone, including tartrate‐resistant acid phosphatase (TRAP) and cathepsin K, that returned to virgin levels upon weaning. Infusion of parathyroid hormone–related peptide (PTHrP), known to be elevated during lactation, induced TRAP activity and cathepsin K expression in osteocytes concurrent with osteocytic remodeling. Conversely, animals lacking the parathyroid hormone type 1 receptor (PTHR1) in osteocytes failed to express TRAP or cathepsin K or to remodel their osteocyte perilacunar matrix during lactation. These studies show that osteocytes remove mineralized matrix through molecular mechanisms similar to those utilized by osteoclasts. © 2012 American Society for Bone and Mineral Research.     

Supraphysiological loading induces osteocyte‐mediated osteoclastogenesis in a novel in vitro model for bone implant loosening

We aimed to develop an in vitro model for bone implant loosening, allowing analysis of biophysical and biological parameters contributing to mechanical instability‐induced osteoclast differentiation and peri‐implant bone loss. MLO‐Y4‐osteocytes were mechanically stimulated for 1 h by fluid shear stress using regimes simulating: (i) supraphysiological loading in the peri‐prosthetic interface (2.9 ± 2.9 Pa, 1 Hz, square wave); (ii) physiologic loading in the cortical bone (0.7 ± 0.7 Pa, 5 Hz, sinusoidal wave); and (iii) stress shielding. Cellular morphological parameters, membrane‐bound RANKL expression, gene expression influencing osteoclast differentiation, nitric oxide release and caspase 3/7‐activity were determined. Either Mouse bone marrow cells were cultured on top of loaded osteocytes or osteocyte‐conditioned medium was added to bone marrow cells. Osteoclast differentiation was assessed after 6 days. We found that osteocytes subjected to supraphysiological loading showed similar morphology and caspase 3/7‐activity compared to simulated physiological loading or stress shielding. Supraphysiological stimulation of osteocytes enhanced osteoclast differentiation by 1.9‐fold compared to physiological loading when cell‐to‐cell contact was permitted. In addition, it enhanced the number of osteoclasts using conditioned medium by 1.7‐fold, membrane‐bound RANKL by 3.3‐fold, and nitric oxide production by 3.2‐fold. The stimulatory effect of supraphysiological loading on membrane‐bound RANKL and nitric oxide production was higher than that achieved by stress shielding. In conclusion, the in vitro model developed recapitulated the catabolic biological situation in the peri‐prosthetic interface during instability that is associated with osteoclast differentiation and enhanced RANKL expression. The model thus provides a platform for pre‐clinical testing of pharmacological interventions with potential to stop instability‐induced bone implant loosening. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1425–1434, 2018.

     

Osteocytes Acidify Their Microenvironment in Response to PTHrP In Vitro and in Lactating Mice In Vivo

Osteocytes appear to mobilize calcium within minutes in response to PTH injections; we have previously shown that osteocytes remove their perilacunar matrix during lactation through activation of the PTH type 1 receptor. Mechanisms utilized by osteocytes to mobilize calcium are unknown but we hypothesized that the molecular components may be similar to those used by osteoclasts. Here we show, using IDG‐SW3 cells that ATP6V0D2, an essential component of vacuolar ATPase in osteoclasts, and other genes associated with osteoclastic bone resorption, increase with osteoblast to osteocyte differentiation. Furthermore, PTHrP increases ATP6V0D2 expression and induces proton generation by primary osteocytes, which is blocked by bafilomycin, a vacuolar ATPase inhibitor. These in vitro proton measurements raised the question of osteocyte viability in an acidic environment. Interestingly, osteocytes, showed enhanced viability at pH as low as 5 compared to osteoblasts and fibroblasts in vitro. To study in vivo acidification by osteocytes, virgin and lactating CD1 mice on a low calcium diet were injected with the pH indicator dye, acridine orange, and their osteocyte lacuno‐canalicular system imaged by confocal microscopy. Lower pH was observed in lactating compared to virgin animals. In addition, a novel transgenic mouse line with a topaz variant of green fluorescent protein (GFPtpz)‐tagged collagen α2(I) chain was used. Instead of the expected reduction in GFP‐fluorescence only in the perilacunar matrix, reduced fluorescence was observed in the entire bone matrix of lactating mice. Based on our experiments showing quenching of GFP in vitro, we propose that the observed reduction in GFP fluorescence in lactating mice is due to quenching of GFP by the acidic pH generated by osteocytes. Together these findings provide novel mechanistic insight into how osteocytes remove calcium from their perilacunar/pericanalicular matrices through active acidification of their microenvironment and show that osteocytes, like osteoclasts, are resistant to the negative effects of acid on viability. © 2017 American Society for Bone and Mineral Research.     

Focal adhesion protein Kindlin-2 regulates bone homeostasis in mice

Our recent studies demonstrate that the focal adhesion protein Kindlin-2 is critical for chondrogenesis and early skeletal development. Here, we show that deleting Kindlin-2 from osteoblasts using the 2.3-kb mouse Col1 a1-Cre transgene minimally impacts bone mass in mice, but deleting Kindlin-2 using the 10-kb mouse Dmp1-Cre transgene, which targets osteocytes and mature osteoblasts, results in striking osteopenia in mice. Kindlin-2 loss reduces the osteoblastic population but increases the osteoclastic and adipocytic populations in the bone microenvironment. Kindlin-2 loss upregulates sclerostin in osteocytes,downregulates β-catenin in osteoblasts, and inhibits osteoblast formation and differentiation in vitro and in vivo. Upregulation ofβ-catenin in the mutant cells reverses the osteopenia induced by Kindlin-2 deficiency. Kindlin-2 loss additionally increases the expression of RANKL in osteocytes and increases osteoclast formation and bone resorption. Kindlin-2 deletion in osteocytes promotes osteoclast formation in osteocyte/bone marrow monocyte cocultures, which is significantly blocked by an anti-RANKLneutralizing antibody. Finally, Kindlin-2 loss increases osteocyte apoptosis and impairs osteocyte spreading and dendrite formation.Thus, we demonstrate an important role of Kindlin-2 in the regulation of bone homeostasis and provide a potential target for the treatment of metabolic bone diseases.     

Osteocyte and bone structure

The osteocyte is the most abundant cell type of bone. There are approximately 10 times as many osteocytes as osteoblasts in adult human bone, and the number of osteoclasts is only a fraction of the number of osteoblasts. Our current knowledge of the role of osteocytes in bone metabolism is far behind our insight into the properties and functions of the osteoblasts and osteoclasts. However, the striking structural design of bone predicts an important role for osteocytes in determining bone structure. Over the past several years, the role of osteocytes as the professional mechanosensory cells of bone, and the lacunocanalicular porosity as the structure that mediates mechanosensing have become clear. Strain-derived flow of interstitial fluid through this porosity seems to mechanically activate the osteocytes, as well as ensure transport of cell signaling molecules, nutrients, and waste products. This concept explains local bone gain and loss—as well as remodeling in response to fatigue damage—as processes supervised by mechanosensitive osteocytes. Alignment during remodeling seems to occur as a result of the osteocyte’s sensing different canalicular flow patterns around the cutting cone and reversal zone during loading, therefore determining the bone’s structure.     

The Role of Osteocytes in Age-Related Bone Loss

The decrease in bone mass and strength during aging has multiple causes. Osteocytes are long-lived cells within the bone matrix that perform a variety of functions, including the control of bone remodeling. Because of their longevity, osteocytes are more likely than osteoclasts or osteoblasts to accumulate molecular damage over time. Osteocytes utilize quality-control pathways like autophagy to remove damaged organelles and macromolecules, and thereby maintain function. When the damage is excessive, cell death pathways such as apoptosis minimize the impact of potential osteocyte dysfunction on the skeleton. The goal of this review is to discuss how dysregulation of these pathways in osteocytes may contribute to the decline in bone mass and strength with age.     

Apoptotic Osteocytes Induce RANKL Production in Bystanders via Purinergic Signaling and Activation of Pannexin Channels

Localized apoptosis of osteocytes, the tissue-resident cells within bone, occurs with fatigue microdamage and activates bone resorption. Osteoclasts appear to target and remove dying osteocytes, resorbing damaged bone matrix as well. Osteocyte apoptosis similarly activates bone resorption with estrogen loss and in disuse. Apoptotic osteocytes trigger viable neighbor (ie, bystander) osteocytes to produce RANKL, the cytokine required for osteoclast activation. Signals from apoptotic osteocytes that trigger this bystander RANKL expression remain obscure. Studying signaling among osteocytes has been hampered by lack of in vitro systems that model the limited communication among osteocytes in vivo (ie, via gap junctions on cell processes and/or paracrine signals through thin pericellular fluid spaces around osteocytes). Here, we used a novel multiscale fluidic device (the Macro-micro-nano, or Mμn) that reproduces these key anatomical features. Osteocytes in discrete compartments of the device communicate only via these limited pathways, which allows assessment of their roles in triggering osteocytes RANKL expression. Apoptosis of MLOY-4 osteocytes in the Mμn device caused increased osteocyte RANKL expression in the neighboring compartment, consistent with in vivo findings. This RANKL upregulation in bystander osteocytes was prevented by blocking Pannexin 1 channels as well as its ATP receptor. ATP alone caused comparable RANKL upregulation in bystander osteocytes. Finally, blocking Connexin 43 gap junctions did not abolish osteocyte RANKL upregulation, but did alter the distribution of RANKL expressing bystander osteocytes. These findings point to extracellular ATP, released from apoptotic osteocytes via Panx1 channels, as a major signal for triggering bystander osteocyte RANKL expression and activating bone remodeling. © 2020 American Society for Bone and Mineral Research.     

Osteocyte Apoptosis Controls Activation of Intracortical Resorption in Response to Bone Fatigue

Osteocyte apoptosis is spatially and temporally linked to bone fatigue‐induced microdamage and to subsequent intracortical remodeling. Specifically, osteocytes surrounding fatigue microcracks in bone undergo apoptosis, and those regions containing apoptotic osteocytes co‐localize exactly with areas subsequently resorbed by osteoclasts. Here we tested the hypothesis that osteocyte apoptosis is a key controlling step in the activation and/or targeting of osteoclastic resorption after bone fatigue. We carried out in vivo fatigue loading of ulna from 4‐ to 5‐mo‐old Sprague‐Dawley rats treated with an apoptosis inhibitor (the pan‐caspase inhibitor Q‐VD‐OPh) or with vehicle. Intracortical bone remodeling and osteocyte apoptosis were quantitatively assessed by standard histomorphometric techniques on day 14 after fatigue. Continuous exposure to Q‐VD‐OPh completely blocked both fatigue‐induced apoptosis and the activation of osteoclastic resorption, whereas short‐term caspase inhibition during only the first 2 days after fatigue resulted in >50% reductions in both osteocyte apoptosis and bone resorption. These results (1) show that osteocyte apoptosis is necessary to initiate intracortical bone remodeling in response to fatigue microdamage, (2) indicate a possible dose‐response relationship between the two processes, and (3) suggest that early apoptotic events after fatigue‐induced microdamage may play a substantial role in determining the subsequent course of tissue remodeling.     

Isolated chick osteocytes stimulate formation and bone-resorbing activity of osteoclast-like cells

Several possible roles have been proposed for osteocytes in bone metabolism, but the precise functions of these cells are still obscure. In this study, we examined the effects of osteocytes on osteoclast formation, using isolated osteocytes from 16-day-old chick embryonic parietal bones and mouse hemopoietic blast cells from spleen cells obtained from 5-fluorouracil-treated mice. Purity of the isolated osteocytes was more than 95%, and their osteocytic phenotypes—such as those having a typical stellate shape and being immunoreactive for osteocyte-specific monoclonal antibody OB 7.3, the nonproliferative phenotype, and those negative for alkaline phosphatase (ALP) activity—were retained during 3 days of culture. When hemopoietic blast cells were cocultured with the isolated osteocytes, the number of osteoclastic tartrate-resistant acid phosphatase (TRAP)-positive multinucleate cells (MNCs) from the blast cells increased even in the absence of any osteotropic factors. This stimulatory effect depended on the number of osteocytes added, and was consistent with that obtained with bone fragments freed of nonosteocytic cells by stripping the periosteum. Conditioned medium (CM) of isolated osteocytes also had stimulatory effects on both formation and bone-resorbing activity of the MNCs, indicating that osteocytes increased osteoclastic development, at least in part, via their production of some soluble factor(s). This osteoclast-inducing activity in the osteocyte CM was observed in the fraction that eluted at around 0.3 M NaCl from a mono-Q anion-exchange column, and this elution profile was similar to that of cell lysates of freshly isolated osteocytes and of stripped bone fragments, indicating the existence of such osteoclastogenesis factor(s) in vivo. Finally, both the osteocyte CM and the active fraction of mono-Q chromatography increased the pit area on dentine slices in the cultures of unfractionated mouse bone cells as well. Thus, osteocytes may play a role in the physiological processes of osteoclastic bone resorption.     

Effects of Type 1 Diabetes on Osteoblasts,Osteocytes, and Osteoclasts

Purpose of Review

To describe the effects of type 1 diabetes on bone cells.

Recent findings

Type 1 diabetes (T1D) is associated with low bone mineral density, increased risk of fractures, and poor fracture healing. Its effects on the skeleton were primarily attributed to impaired bone formation, but recent data suggests that bone remodeling and resorption are also compromised. The hyperglycemic and inflammatory environment associated with T1D impacts osteoblasts, osteocytes, and osteoclasts. The mechanisms involved are complex; insulinopenia, pro-inflammatory cytokine production, and alterations in gene expression are a few of the contributing factors leading to poor osteoblast activity and survival and, therefore, poor bone formation. In addition, the observed sclerostin level increase accompanied by decreased osteocyte number and enhanced osteoclast activity in T1D results in uncoupling of bone remodeling.

Summary

T1D negatively impacts osteoblasts and osteocytes, whereas its effects on osteoclasts are not well characterized, although the limited studies available indicate increased osteoclast activity, favoring bone resorption.

     

Osteocyte apoptosis regulates osteoclast precursor adhesion via osteocytic IL-6 secretion and endothelial ICAM-1 expression

Osteocyte apoptosis precedes osteoclast resorption, and may act as a critical signal to trigger bone remodeling. While osteoclast precursors are known to travel via the circulation, the specific mechanisms by which they accumulate at remodeling sites are unclear. We hypothesized that osteocyte apoptosis mediates osteoclast precursor adhesion to vascular endothelium by regulating osteocytic secretion of IL-6 and soluble IL-6 receptor (sIL-6R) to promote endothelial ICAM-1 expression. We found that conditioned media from TNF-α-induced apoptotic MLO-Y4 osteocytes promoted RAW264.7 osteoclast precursor adhesion onto D4T endothelial cells (P < 0.05). Blocking osteocyte apoptosis with a pan-caspase inhibitor (ZVAD-FMK) reduced osteoclast precursor adhesion to baseline levels (P < 0.001). Endothelial cells treated with apoptotic osteocyte conditioned media had elevated surface expression of ICAM-1 (P < 0.05), and blocking ICAM-1 abolished apoptosis-induced osteoclast precursor adhesion. Apoptotic osteocyte conditioned media contained more IL-6 (P < 0.05) and sIL-6R (P < 0.05) than non-apoptotic osteocyte conditioned media. When added exogenously, both IL-6 and sIL-6R were required for endothelial activation, and blocking IL-6 reduced apoptosis-induced osteoclast precursor adhesion to baseline levels (P < 0.05). Therefore, we conclude that osteocyte apoptosis can promote osteoclast precursor adhesion to endothelial cells via ICAM-1; this is likely through increased osteocytic IL-6 and sIL-6R secretion, both of which are indispensible to endothelial activation.     

Osteocyte apoptosis is induced by weightlessness in mice and precedes osteoclast recruitment and bone loss.

Mechanical stimulation of cultured osteocytic cells attenuates their apoptosis. We report here that, conversely, reduced mechanical forces in the murine model of unloading by tail suspension increases the prevalence of osteocyte apoptosis, followed by bone resorption and loss of mineral and strength. INTRODUCTION: Mechanical loading is critical for the maintenance of bone mass; weightlessness, as with reduced physical activity in old age, bed rest, or space flight, invariably leads to bone loss. However, the cellular and molecular mechanisms responsible for these phenomena are poorly understood. Based on our earlier findings that physiologic levels of mechanical strain prevent apoptosis of osteocytic cells in vitro, we examined here whether, conversely, reduced mechanical forces increase the prevalence of osteocyte apoptosis in vivo and whether this event is linked to bone loss. MATERIALS AND METHODS: Swiss Webster mice or OG2-11beta-hydroxysteroid dehydrogenase type 2 (OG2-11beta-HSD2) transgenic mice and wildtype littermates were tail-suspended or kept under ambulatory conditions. Static and dynamic histomorphometry and osteocyte and osteoblast apoptosis by in situ end-labeling (ISEL) were assessed in lumbar vertebra; spinal BMD was measured by DXA; and bone strength was measured by vertebral compression. RESULTS: We show that within 3 days of tail suspension, mice exhibited an increased incidence of osteocyte apoptosis in both trabecular and cortical bone. This change was followed 2 weeks later by increased osteoclast number and cortical porosity, reduced trabecular and cortical width, and decreased spinal BMD and vertebral strength. Importantly, whereas in ambulatory animals, apoptotic osteocytes were randomly distributed, in unloaded mice, apoptotic osteocytes were preferentially sequestered in endosteal cortical bone--the site that was subsequently resorbed. The effect of unloading on osteocyte apoptosis and bone resorption was reproduced in transgenic mice in which osteocytes are refractory to glucocorticoid action, indicating that stress-induced hypercortisolemia cannot account for these effects. CONCLUSIONS: We conclude that diminished mechanical forces eliminate signals that maintain osteocyte viability, thereby leading to apoptosis. Dying osteocytes in turn become the beacons for osteoclast recruitment to the vicinity and the resulting increase in bone resorption and bone loss.     

Effects of PTH on osteocyte function

Osteocytes are ideally positioned to detect and respond to mechanical and hormonal stimuli and to coordinate the function of osteoblasts and osteoclasts. However, evidence supporting the involvement of osteocytes in specific aspects of skeletal biology has been limited mainly due to the lack of suitable experimental approaches. Few crucial advances in the field in the past several years have markedly increased our understanding of the function of osteocytes. The development of osteocytic cell lines initiated a plethora of in vitro studies that have provided insights into the unique biology of osteocytes and continue to generate novel hypotheses. Genetic approaches using promoter fragments that direct gene expression to osteocytes allowed the generation of mice with gain or loss of function of particular genes revealing their role in osteocyte function. Furthermore, evidence that Sost/sclerostin is expressed primarily in osteocytes and inhibits bone formation by osteoblasts, fueled research attempting to identify regulators of this gene as well as other osteocyte products that impact the function of osteoblasts and osteoclasts. The discovery that parathyroid hormone (PTH), a central regulator of bone homeostasis, inhibits sclerostin expression generated a cascade of studies that revealed that osteocytes are crucial target cells of the actions of PTH. This review highlights these investigations and discusses their significance for advancing our understanding of the mechanisms by which osteocytes regulate bone homeostasis and for developing therapies for bone diseases targeting osteocytes. This article is part of a Special Issue entitled "The Osteocyte".     

Upregulation of osteopontin by osteocytes deprived of mechanical loading or oxygen.

The pathway(s) by which disuse is transduced into locally mediated osteoclastic resorption remain unknown. We found that both acute disuse (in vivo) and direct hypoxia (in vitro) induced rapid upregulation of OPN expression by osteocytes. Within the context of OPN's role in osteoclast migration and attachment, hypoxia-induced osteocyte OPN expression may serve to mediate disuse-induced bone resorption. INTRODUCTION: We have recently reported that disuse induces osteocyte hypoxia. Because hypoxia upregulates osteopontin (OPN) in nonconnective tissue cells, we hypothesized that both disuse and hypoxia would rapidly elevate expression of OPN by osteocytes. MATERIALS AND METHODS: The response of osteocytes to 24 h of disuse was explored by isolating the left ulna diaphysis of adult male turkeys from loading (n = 5). Cortical osteocytes staining positive for OPN were determined using immunohistochemistry and confocal microscopy. In vitro experiments were performed to determine if OPN expression was altered in MLO-Y4 osteocytes by direct hypoxia (3, 6, 24, and 48 h) or hypoxia (3 and 24 h) followed by 24 h of reoxygenation. A final in vitro experiment explored the potential of protein kinase C (PKC) to regulate hypoxia-induced osteocyte OPN mRNA alterations. RESULTS: We found that 24 h of disuse significantly elevated osteocyte OPN expression in vivo (145% versus intact bones; p = 0.02). We confirmed this finding in vitro, by observing rapid and significant upregulation of OPN protein expression after 24 and 48 h of hypoxia. Whereas 24 h of reoxygenation after 3 h of hypoxia restored normal osteocyte OPN expression levels, 24 h of reoxygenation after 24 h of hypoxia did not mitigate elevated osteocyte OPN expression. Finally, preliminary inhibitor studies suggested that PKC serves as a potent upstream regulator of hypoxia-induced osteocyte OPN expression. CONCLUSIONS: Given the documented roles of OPN as a mediator of environmental stress (e.g., hypoxia), an osteoclast chemotaxant, and a modulator of osteoclastic attachment to bone, we speculate that hypoxia-induced osteocyte OPN expression may serve to mediate disuse-induced osteoclastic resorption. Furthermore, it seems that a brief window of time exists in which reoxygenation (as might be achieved by reloading bone) can serve to inhibit this pathway.