Apoptotic osteocytes and the control of targeted bone resorption

Studies from the 1950s and 1960s already recognize the fact that osteocytes, although long living cells, die, as evidenced by accumulation of osteocytic lacunae devoid of cells. More recently, it was demonstrated that these cells die by apoptosis. The rate of osteocyte apoptosis is regulated by the age of the bone, as well as by systemic hormones, local growth factors, cytokines, pharmacological agents, and mechanical forces. Apoptotic osteocytes, in turn, recruit osteoclasts to initiate targeted bone resorption. This results in the removal of “dead” bone and may improve the mechanical properties of the skeleton. However, the molecular regulators of osteocyte survival and targeted bone remodeling are not completely known. In this review, the current knowledge on the molecular mechanism that lead to osteocyte death or survival, and the signals that mediate targeted bone resorption is discussed

RAGE Signaling in Skeletal Biology

PURPOSE OF REVIEW: The receptor for advanced glycation end products (RAGE) and several of its ligands have been implicated in the onset and progression of pathologies associated with aging, chronic inflammation, and cellular stress. In particular, the role of RAGE and its ligands in bone tissue during both physiological and pathological conditions has been investigated. However, the extent to which RAGE signaling regulates bone homeostasis and disease onset remains unclear. Further, RAGE effects in the different bone cells and whether these effects are cell-type specific is unknown. The objective of the current review is to describe the literature over RAGE signaling in skeletal biology as well as discuss the clinical potential of RAGE as a diagnostic and/or therapeutic target in bone disease. RECENT FINDINGS: The role of RAGE and its ligands during skeletal homeostasis, tissue repair, and disease onset/progression is beginning to be uncovered. For example, detrimental effects of the RAGE ligands, advanced glycation end products (AGEs), have been identified for osteoblast viability/activity, while others have observed that low level AGE exposure stimulates osteoblast autophagy, which subsequently promotes viability and function. Similar findings have been reported with HMGB1, another RAGE ligand, in which high levels of the ligand are associated with osteoblast/osteocyte apoptosis, whereas low level/short-term administration stimulates osteoblast differentiation/bone formation and promotes fracture healing. Additionally, elevated levels of several RAGE ligands (AGEs, HMGB1, S100 proteins) induce osteoblast/osteocyte apoptosis and stimulate cytokine production, which is associated with increased osteoclast differentiation/activity. Conversely, direct RAGE-ligand exposure in osteoclasts may have inhibitory effects. These observations support a conclusion that elevated bone resorption observed in conditions of high circulating ligands and RAGE expression are due to actions on osteoblasts/osteocytes rather than direct actions on osteoclasts, although additional work is required to substantiate the observations. Recent studies have demonstrated that RAGE and its ligands play an important physiological role in the regulation of skeletal development, homeostasis, and repair/regeneration. Conversely, elevated levels of RAGE and its ligands are clearly related with various diseases associated with increased bone loss and fragility. However, despite the recent advancements in the field, many questions regarding RAGE and its ligands in skeletal biology remain unanswered

Connexins and pannexins in the skeleton: gap junctions, hemichannels and more

Regulation of bone homeostasis depends on the concerted actions of bone-forming osteoblasts and bone-resorbing osteoclasts, controlled by osteocytes, cells derived from osteoblasts surrounded by bone matrix. The control of differentiation, viability and function of bone cells relies on the presence of connexins. Connexin43 regulates the expression of genes required for osteoblast and osteoclast differentiation directly or by changing the levels of osteocytic genes, and connexin45 may oppose connexin43 actions in osteoblastic cells. Connexin37 is required for osteoclast differentiation and its deletion results in increased bone mass. Less is known on the role of connexins in cartilage, ligaments and tendons. Connexin43, connexin45, connexin32, connexin46 and connexin29 are expressed in chondrocytes, while connexin43 and connexin32 are expressed in ligaments and tendons. Similarly, although the expression of pannexin1, pannexin2 and pannexin3 has been demonstrated in bone and cartilage cells, their function in these tissues is not fully understood

β-arrestin/connexin 43 complex anchors ERKs outside the nucleus: a pre-requisite for bisphosphonate anti-apoptotic effect mediated by CX43/ERK in osteocytes

Bisphosphonates (BPs) anti-fracture ef cacy may be due in part to inhibition of osteocyte apoptosis. This effect requires opening of con- nexin (Cx) 43 hemichannels and phosphoryla- tion of the extracellular signal regulated kinases (ERKs). However, unlike ERK activation by other stimuli, the Cx43/ERK pathway activated by BPs does not result in nuclear ERK accumulation. In- stead, the anti-apoptotic effect of BPs depends on phosphorylation of cytoplasmic ERK targets and is abolished by forced nuclear retention of ERKs. We now report that ERKs and the scaf- folding protein β-arrestin co-immuno-precipitate with Cx43 in MLO-Y4 osteocytic cells and that the BP alendronate increases this association. Moreover, ERK2 fused to red uorescent pro- tein (ERK2-RFP) co-localizes with Cx43 fused to green uorescent protein outside the nucleus in cells untreated or treated with alendronate. Alendronate does not induce ERK nuclear ac- cumulation in cells transfected with wild type β-arrestin (wtARR) or vector control, whereas it does in cells expressing a dominant nega- tive β-arrestin mutant (dnARR) consisting of the β-arrestin-clathrin binding domain that com- petes with endogenous β-arrestin for binding to clathrin. Alendronate activates ERKs in dnARR- transfected cells as effectively as in cells trans- fected with wtARR, demonstrating that dnARR only interferes with subcellular localization but not with activation of ERKs by BPs. Further, whereas alendronate inhibits apoptosis in cells expressing wtARR or vector control, it is inef- fective in cells expressing dnARR. Thus, BPs induce the formation of a complex comprising Cx43, β-arrestin, and clathrin, which directs ERKs outside the nucleus and is indispensable for osteocyte survival induced by BPs

Cx43 and mechanotransduction in bone

Bone adaptation to changes in mechanical stimuli occurs by adjusting bone formation and resorption by osteoblasts and osteoclasts, to maintain optimal bone mass. Osteocytes coordinate the actions of these cells on the bone surface by sensing mechanical forces and producing cytokines that increase or prevent osteoblast and osteoclast differentiation and function. Channels formed by connexins (Cxs) and, in particular, connexin 43 (Cx43) in osteoblasts and osteocytes are central part of this mechanism to control bone mass. Cx43 hemichannels are opened by fluid flow and mediate the anti-apoptotic effect of mechanical stimulation in vitro, suggesting that Cx43 participates in mechanotransduction. However, mice lacking Cx43 in osteoblasts and/or osteocytes show an increased anabolic response to loading and decreased catabolic response to unloading. This evidence suggests that Cx43 channels expressed in osteoblastic cells are not required for the response to mechanical stimulation, but mediate the consequence of lack thereof. The molecular basis of these unexpected responses to mechanical stimulation is currently under investigation

microRNAs and connexins in bone: interaction and mechanisms of delivery

PURPOSE OF REVIEW: To describe the current knowledge on the cross-talk between connexins and microRNAs (miRs) in bone cells. RECENT FINDINGS: Connexins play a crucial role on bone development and maintenance, and disruptions in their abundance or localization can affect how bone perceives and responds to mechanical, hormonal, and pharmacological stimuli. Connexin expression can be modified by miRs, which modulate connexin mRNA and protein levels. Recently, different manners by which miRs and connexins can interact in bone have been identified, including mechanisms that mediate miR exchange between cells in direct contact through gap junctions, or between distant cells via extracellular vesicles (EVs). SUMMARY: We bring to light the relationship between miRs and connexins in bone tissue, with special focus on regulatory effects of miRs and connexins on gene expression, as well as the mechanisms that mediate miR exchange between cells in direct contact through gap junctions, or between distant cells via EVs

Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner

Oats contain unique bioactive compounds known as avenanthramides (AVAs) with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin) in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT) and Nrf2 Knockout (KO) osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast apoptosis; further, these regulatory actions are independent of Nrf2

Prevention of glucocorticoid induced-apoptosis of osteoblasts and osteocytes by protecting against endoplasmic reticulum (ER) stress in vitro and in vivo in female mice

Endoplasmic reticulum (ER) stress is associated with increased reactive oxygen species (ROS), results from accumulation of misfolded/unfolded proteins, and can trigger apoptosis. ER stress is alleviated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which inhibits protein translation allowing the ER to recover, thus promoting cell viability. We investigated whether osteoblastic cell apoptosis induced by glucocorticoids (GCs) is due to induction of ROS/ER stress and whether inhibition of eIF2α dephosphorylation promotes survival opposing the deleterious effects of GC in vitro and in vivo. Apoptosis of osteocytic MLO-Y4 and osteoblastic OB-6 cells induced by dexamethasone was abolished by ROS inhibitors. Like GC, the ER stress inducing agents brefeldin A and tunicamycin induced osteoblastic cell apoptosis. Salubrinal or guanabenz, specific inhibitors of eIF2α dephosphorylation, blocked apoptosis induced by either GC or ER stress inducers. Moreover, GC markedly decreased mineralization in OB-6 cells or primary osteoblasts; and salubrinal or guanabenz increased mineralization and prevented the inhibitory effect of GC. Furthermore, salubrinal (1 mg/kg/day) abolished osteoblast and osteocyte apoptosis in cancellous and cortical bone and partially prevented the loss of BMD at all sites and the decreased vertebral cancellous bone formation induced by treatment with prednisolone for 28 days (1.4 mg/kg/day). We conclude that part of the pro-apoptotic actions of GC on osteoblastic cells is mediated through ER stress, and that inhibition of eIF2α dephosphorylation protects from GC-induced apoptosis of osteoblasts and osteocytes in vitro and in vivo and from the deleterious effects of GC on the skeleton

From inside your bones: Osteocytic signaling pathways as therapeutic targets for bone fragility

Osteocytes are differentiated osteoblasts that become surrounded by matrix during the process of bone formation. The acquisition of the osteocyte phenotype is achieved by profound modifications in gene expression that confer adaptation to the changing cellular functions and constitute the molecular signature of osteocytes. The levels of expression of genes characteristic of osteoblasts is altered; and the expression of genes/proteins that impart dendritic cellular morphology, regulate matrix mineralization, and control the function of bone surface cells, is orderly modulated during osteocytogenesis. The discovery of human mutations of osteocytic genes had contributed to a large extent to reveal the role of osteocytes in bone homeostasis. Osteocytes are targets of mechanical force imposed to the skeleton and also play a critical role in integrating mechanosensory pathways with the action of hormones, thereby leading to the orchestrated response of bone to environmental cues. Current, novel therapeutic approaches harness this accumulating knowledge by targeting osteocytic signaling pathways and messengers to improve skeletal health

Osteocytic connexin 43 is not required for the increase in bone mass induced by intermittent PTH administration in male mice

Objective: To investigate whether osteocytic connexin 43 (Cx43) is required for the bone response to intermittent PTH administration, and whether the connexin is involved in maintaining the bone matrix. Methods: Human PTH(1-34) was injected to adult male mice expressing (Cx43fl/fl) or not osteocytic Cx43 (Cx43fl/fl;DMP1-8kb-Cre) daily (100 μg/kg/d) for 14 days. Results: Cx43fl/fl;DMP1-8kb-Cre mice have no difference in body weight and BMD from 1 to 4 months of age. Intermittent PTH administration increased BMD and BV/TV and induced a similar increase in type I collagen, alkaline phosphatase, runx2, osteocalcin, and bone sialoprotein expression in mice from both genotypes. On the other hand, osteocytic deletion of Cx43 did not alter mRNA levels of glycosaminoglycans, proteoglycans, collagens and osteoblast-related genes. In addition, expression of collagens assessed by immunohistochemistry was not affected by deleting osteocytic Cx43. However, PTH administration increased type II collagen only in Cx43fl/fl control mice, whereas hormone increased type I collagen expression only in Cx43fl/fl;DMP1-8kb-Cre mice. Furthermore, PTH increased maturity of collagen fibers in control, but not in Cx43-deficient mice. Conclusion: Expression of Cx43 in osteocytes is dispensable for bone anabolism induced by intermittent PTH administration; but it can modulate, at least in part, the effect of PTH on the bone matrix environment