The Emerging Role of Osteocytes in Cancer in Bone

Advances in the last decade have established the osteocyte, the most abundant cell in bone, as a dynamic and multifunctional cell capable of controlling bone homeostasis by regulating the function of both osteoblasts and osteoclasts. In addition, accumulating evidence demonstrates that osteocyte function is altered in several skeletal disorders, and targeting osteocytes and their derived factors improves skeletal health. Despite the remarkable progress in our understanding of osteocyte biology, there has been a paucity of information regarding the role of osteocytes in the progression of cancer in bone. Exciting, recent discoveries suggest that tumor cells communicate with osteocytes to generate a microenvironment that supports the growth and survival of cancer cells and stimulates bone destruction. This review features these novel findings and discussions regarding the impact of chemotherapy on osteocyte function and the potential of targeting osteocytes for the treatment of cancer in bone. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research

Aplidin (plitidepsin) is a novel anti-myeloma agent with potent anti-resorptive activity mediated by direct effects on osteoclasts

Despite recent progress in its treatment, Multiple Myeloma (MM) remains incurable and its associated bone disease persists even after complete remission. Thus, identification of new therapeutic agents that simultaneously suppress MM growth and protect bone is an unmet need. Herein, we examined the effects of Aplidin, a novel anti-cancer marine-derived compound, on MM and bone cells. In vitro, Aplidin potently inhibited MM cell growth and induced apoptosis, effects that were enhanced by dexamethasone (Dex) and bortezomib (Btz). Aplidin modestly reduced osteocyte/osteoblast viability and decreased osteoblast mineralization, effects that were enhanced by Dex and partially prevented by Btz. Further, Aplidin markedly decreased osteoclast precursor numbers and differentiation, and reduced mature osteoclast number and resorption activity. Moreover, Aplidin reduced Dex-induced osteoclast differentiation and further decreased osteoclast number when combined with Btz. Lastly, Aplidin alone, or suboptimal doses of Aplidin combined with Dex or Btz, decreased tumor growth and bone resorption in ex vivo bone organ cultures that reproduce the 3D-organization and the cellular diversity of the MM/bone marrow niche. These results demonstrate that Aplidin has potent anti-myeloma and anti-resorptive properties, and enhances proteasome inhibitors blockade of MM growth and bone destruction

MLO-Y4 osteocytic cell clones express distinct gene expression patterns characteristic of different stages of osteocyte differentiation

Osteocytes are the most abundant bone cell and are formed when osteoblasts become embedded in the bone matrix. Through changes in gene expression and paracrine effects, osteocytes regulate the number of osteoblasts, bone forming cells, and osteoclasts, bone resorbing cells, which are needed to maintain bone mass. MLO-Y4 is the better characterized osteocytic cell line; however, lacks expression of sclerostin, the product of the SOST gene, which is fundamental for osteocyte function and blocks bone formation. With the objective to isolate MLO-Y4 clones with different gene expression profiles, we performed cultures at very low density of MLO-Y4 cells stably transfected with nuclear green fluorescent protein (MLO-nGFP). Cell morphology was visualized under a fluorescence microscope. Once the cells reached 80% confluency, RNA was extracted and quantitative real time PCR was performed. Clones exhibit different sizes and morphology, with some cells showing a spindle-like shape and others with abundant projections and a star-like shape. Gene expression also differed among clones. However, none of the clones examined expressed SOST. We conclude that the MLO-nGFP clones constitute a useful tool to study osteocyte differentiation and the role of osteocytes in the control of bone formation and resorption in vitro

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

Public Health Research Priorities to Address US Human Trafficking

In this Perspective, HEAL Trafficking, the nation\u27s leading public health anti-trafficking organization maps out a national research agenda to tackle the problem of human trafficking. Given the paucity of research on trafficking, HEAL Trafficking engaged its membership in a consensus development process throughout 2016 to develop its national research agenda. HEAL Trafficking proposes five priorities that public health researchers should focus on in the decade ahead to make meaningful progress on preventing and responding to human trafficking in the Unites States

Reversal of loss of bone mass in old mice treated with mefloquine

Aging is accompanied by imbalanced bone remodeling, elevated osteocyte apoptosis, and decreased bone mass and mechanical properties; and improved pharmacologic approaches to counteract bone deterioration with aging are needed. We examined herein the effect of mefloquine, a drug used to treat malaria and systemic lupus erythematosus and shown to ameliorate bone loss in glucocorticoid-treated patients, on bone mass and mechanical properties in young and old mice. Young 3.5-month-old and old 21-month-old female C57BL/6 mice received daily injections of 5 mg/kg/day mefloquine for 14 days. Aging resulted in the expected changes in bone volume and mechanical properties. In old mice mefloquine administration reversed the lower vertebral cancellous bone volume and bone formation; and had modest effects on cortical bone volume, thickness, and moment of inertia. Mefloquine administration did not change the levels of the circulating bone formation markers P1NP or alkaline phosphatase, whereas levels of the resorption marker CTX showed trends towards increase with mefloquine treatment. In addition, and as expected, aging bones exhibited an accumulation of active caspase3-expressing osteocytes and higher expression of apoptosis-related genes compared to young mice, which were not altered by mefloquine administration at either age. In young animals, mefloquine induced higher periosteal bone formation, but lower endocortical bone formation. Further, osteoclast numbers were higher on the endocortical bone surface and circulating CTX levels were increased, in mefloquine- compared to vehicle-treated young mice. Consistent with this, addition of mefloquine to bone marrow cells isolated from young mice led to increased osteoclastic gene expression and a tendency towards increased osteoclast numbers in vitro. Taken together our findings identify the age and bone-site specific skeletal effects of mefloquine. Further, our results highlight a beneficial effect of mefloquine administration on vertebral cancellous bone mass in old animals, raising the possibility of using this pharmacologic inhibitor to preserve skeletal health with aging

Cx43 Overexpression in Osteocytes Prevents Osteocyte Apoptosis and Preserves Cortical Bone Quality in Aging Mice

Young, skeletally mature mice lacking Cx43 in osteocytes exhibit increased osteocyte apoptosis and decreased bone strength, resembling the phenotype of old mice. Further, the expression of Cx43 in bone decreases with age, suggesting a contribution of reduced Cx43 levels to the age-related changes in the skeleton. We report herein that Cx43 overexpression in osteocytes achieved by using the DMP1-8kb promoter (Cx43OT mice) attenuates the skeletal cortical but not trabecular bone phenotype of aged, 14-month-old mice. The percentage of Cx43-expressing osteocytes was higher in Cx43OT mice, whereas the percentage of Cx43-positive osteoblasts remained similar to wild-type (WT) littermate control mice. The percentage of apoptotic osteocytes and osteoblasts was increased in aged WT mice compared with skeletally mature, 6-month-old WT mice, and the percentage of apoptotic osteocytes, but not osteoblasts, was decreased in age-matched Cx43OT mice. Aged WT mice exhibited decreased bone formation and increased bone resorption as quantified by histomorphometric analysis and circulating markers compared with skeletally mature mice. Further, aged WT mice exhibited the expected decrease in bone biomechanical structural and material properties compared with young mice. Cx43 overexpression prevented the increase in osteoclasts and decrease in bone formation on the endocortical surfaces and the changes in circulating markers in the aged mice. Moreover, the ability of bone to resist damage was preserved in aged Cx43OT mice both at the structural and material level. All together, these findings suggest that increased Cx43 expression in osteocytes ameliorates age-induced cortical bone changes by preserving osteocyte viability and maintaining bone formation, leading to improved bone strength. © 2018 American Society for Bone and Mineral Research

Age- and sex-dependent role of osteocytic pannexin1 on bone and muscle mass and strength

Pannexins (Panxs), glycoproteins that oligomerize to form hemichannels on the cell membrane, are topologically similar to connexins, but do not form cell-to-cell gap junction channels. There are 3 members of the family, 1-3, with Panx1 being the most abundant. All Panxs are expressed in bone, but their role in bone cell biology is not completely understood. We now report that osteocytic Panx1 deletion (Panx1Δot) alters bone mass and strength in female mice. Bone mineral density after reaching skeletal maturity is higher in female Panx1Δot mice than in control Panx1fl/fl mice. Further, osteocytic Panx1 deletion partially prevented aging effects on cortical bone structure and mechanical properties. Young 4-month-old female Panx1Δot mice exhibited increased lean body mass, even though pannexin levels in skeletal muscle were not affected; whereas no difference in lean body mass was detected in male mice. Furthermore, female Panx1-deficient mice exhibited increased muscle mass without changes in strength, whereas Panx1Δot males showed unchanged muscle mass and decreased in vivo maximum plantarflexion torque, indicating reduced muscle strength. Our results suggest that osteocytic Panx1 deletion increases bone mass in young and old female mice and muscle mass in young female mice, but has deleterious effects on muscle strength only in males

Osteocytic miR21 deficiency improves bone strength independent of sex despite having sex divergent effects on osteocyte viability and bone turnover

Osteocytes play a critical role in mediating cell–cell communication and regulating bone homeostasis, and osteocyte apoptosis is associated with increased bone resorption. miR21, an oncogenic microRNA, regulates bone metabolism by acting directly on osteoblasts and osteoclasts, but its role in osteocytes is not clear. Here, we show that osteocytic miR21 deletion has sex‐divergent effects in bone. In females, miR21 deletion reduces osteocyte viability, but suppresses bone turnover. Conversely, in males, miR21 deletion increases osteocyte viability, but stimulates bone turnover and enhances bone structure. Further, miR21 deletion differentially alters osteocyte cytokine production in the two sexes. Interestingly, despite these changes, miR21 deletion increases bone mechanical properties in both sexes, albeit to a greater extent in males. Collectively, our findings suggest that miR21 exerts both sex‐divergent and sex‐equivalent roles in osteocytes, regulating osteocyte viability and altering bone metabolism through paracrine actions on osteoblasts and osteoclasts differentially in males vs females, whereas, influencing bone mechanical properties independent of sex

E4orf1: A Novel Ligand That Improves Glucose Disposal in Cell Culture

Reducing dietary fat intake and excess adiposity, the cornerstones of behavioral treatment of insulin resistance(IR), are marginally successful over the long term. Ad36, a human adenovirus, offers a template to improve IR, independent of dietary fat intake or adiposity. Ad36 increases cellular glucose uptake via a Ras-mediated activation of phosphatidyl inositol 3-kinase(PI3K), and improves hyperglycemia in mice, despite a high-fat diet and without reducing adiposity. Ex-vivo studies suggest that Ad36 improves hyperglycemia in mice by increasing glucose uptake by adipose tissue and skeletal muscle, and by reducing hepatic glucose output. It is impractical to use Ad36 for therapeutic action. Instead, we investigated if the E4orf1 protein of Ad36, mediates its anti-hyperglycemic action. Such a candidate protein may offer an attractive template for therapeutic development. Experiment-1 determined that Ad36 ‘requires’ E4orf1 protein to up-regulate cellular glucose uptake. Ad36 significantly increased glucose uptake in 3T3-L1 preadipocytes, which was abrogated by knocking down E4orf1 with siRNA. Experiment-2 identified E4orf1 as ‘sufficient’ to up-regulate glucose uptake. 3T3-L1 cells that inducibly express E4orf1, increased glucose uptake in an induction-dependent manner, compared to null vector control cells. E4orf1 up-regulated PI3K pathway and increased abundance of Ras–the obligatory molecule in Ad36-induced glucose uptake. Experiment-3: Signaling studies of cells transiently transfected with E4orf1 or a null vector, revealed that E4orf1 may activate Ras/PI3K pathway by binding to Drosophila discs-large(Dlg1) protein. E4orf1 activated total Ras and, particularly the H-Ras isoform. By mutating the PDZ domain binding motif(PBM) of E4orf1, Experiment-4 showed that E4orf1 requires its PBM to increase Ras activation or glucose uptake. Experiment-5: In-vitro, a transient transfection by E4orf1 significantly increased glucose uptake in preadipocytes, adipocytes, or myoblasts, and reduced glucose output by hepatocytes. Thus, the highly attractive anti-hyperglycemic effect of Ad36 is mirrored by E4orf1 protein, which may offer a novel ligand to develop anti-hyperglycemic drugs