Regulation of the Osteocyte Secretome with Aging and Disease.

As the most numerous and long-lived of all bone cells, osteocytes have essential functions in regulating skeletal health. Through the lacunar-canalicular system, secreted proteins from osteocytes can reach cells throughout the bone. Furthermore, the intimate connectivity between the lacunar-canalicular system and the bone vasculature allows for the transport of osteocyte-secreted factors into the circulation to reach the entire body. Local and endocrine osteocyte signaling regulates physiological processes such as bone remodeling, bone mechanoadaptation, and mineral homeostasis. However, these processes are disrupted by impaired osteocyte function induced by aging and disease. Dysfunctional osteocyte signaling is now associated with the pathogenesis of many disorders, including chronic kidney disease, cancer, diabetes mellitus, and periodontitis. In this review, we focus on the targeting of bone and extraskeletal tissues by the osteocyte secretome. In particular, we highlight the secreted osteocyte proteins, which are known to be dysregulated during aging and disease, and their roles during disease progression. We also discuss how therapeutic or genetic targeting of osteocyte-secreted proteins can improve both skeletal and systemic health.

Irisin Mediates Effects on Bone and Fat via αV Integrin Receptors.

Irisin is secreted by muscle, increases with exercise, and mediates certain favorable effects of physical activity. In particular, irisin has been shown to have beneficial effects in adipose tissues, brain, and bone. However, the skeletal response to exercise is less clear, and the receptor for irisin has not been identified. Here we show that irisin binds to proteins of the αV class of integrins, and biophysical studies identify interacting surfaces between irisin and αV/ß5 integrin. Chemical inhibition of the αV integrins blocks signaling and function by irisin in osteocytes and fat cells. Irisin increases both osteocytic survival and production of sclerostin, a local modulator of bone remodeling. Genetic ablation of FNDC5 (or irisin) completely blocks osteocytic osteolysis induced by ovariectomy, preventing bone loss and supporting an important role of irisin in skeletal remodeling. Identification of the irisin receptor should greatly facilitate our understanding of irisin's function in exercise and human health.

Growth of ovarian cancer xenografts causes loss of muscle and bone mass: a new model for the study of cancer cachexia.

BACKGROUND: Cachexia frequently occurs in women with advanced ovarian cancer (OC), along with enhanced inflammation. Despite being responsible for one third of all cancer deaths, cachexia is generally under-studied in OC due to a limited number of pre-clinical animal models. We aimed to address this gap by characterizing the cachectic phenotype in a mouse model of OC. METHODS: Nod SCID gamma mice (n = 6-10) were injected intraperitoneally with 1 × 107 ES-2 human OC cells to mimic disseminated abdominal disease. Muscle size and strength, as well as bone morphometry, were assessed. Tumour-derived effects on muscle fibres were investigated in C2C12 myotube cultures. IL-6 levels were detected in serum and ascites from tumour hosts, as well as in tumour sections. RESULTS: In about 2 weeks, ES-2 cells developed abdominal tumours infiltrating omentum, mesentery, and adjacent organs. The ES-2 tumours caused severe cachexia with marked loss of body weight (-12%, P < 0.01) and ascites accumulation in the peritoneal cavity (4.7 ± 1.5 mL). Skeletal muscles appeared markedly smaller in the tumour-bearing mice (approximately -35%, P < 0.001). Muscle loss was accompanied by fibre atrophy, consistent with reduced muscle cross-sectional area (-34%, P < 0.01) and muscle weakness (-50%, P < 0.001). Body composition assessment by dual-energy X-ray absorptiometry revealed decreased bone mineral density (-8%, P < 0.01) and bone mineral content (-19%, P < 0.01), also consistent with reduced trabecular bone in both femurs and vertebrae, as suggested by micro-CT imaging of bone morphometry. In the ES-2 mouse model, cachexia was also associated with high tumour-derived IL-6 levels in plasma and ascites (26.3 and 279.6 pg/mL, respectively) and with elevated phospho-STAT3 (+274%, P < 0.001), reduced phospho-AKT (-44%, P < 0.001) and decreased mitochondrial proteins, as well as with increased protein ubiquitination (+42%, P < 0.001) and expression of ubiquitin ligases in the skeletal muscle of tumour hosts. Similarly, ES-2 conditioned medium directly induced fibre atrophy in C2C12 mouse myotubes (-16%, P < 0.001), consistent with elevated phospho-STAT3 (+1.4-fold, P < 0.001) and altered mitochondrial homoeostasis and metabolism, while inhibition of the IL-6/STAT3 signalling by means of INCB018424 was sufficient to restore the myotubes size. CONCLUSIONS: Our results suggest that the development of ES-2 OC promotes muscle atrophy in both in vivo and in vitro conditions, accompanied by loss of bone mass, enhanced muscle protein catabolism, abnormal mitochondrial homoeostasis, and elevated IL-6 levels. Therefore, this represents an appropriate model for the study of OC cachexia. Our model will aid in identifying molecular mediators that could be effectively targeted in order to improve muscle wasting associated with OC.

ß-aminoisobutyric Acid, l-BAIBA, Is a Muscle-Derived Osteocyte Survival Factor.

Exercise has beneficial effects on metabolism and on tissues. The exercise-induced muscle factor ß-aminoisobutyric acid (BAIBA) plays a critical role in the browning of white fat and in insulin resistance. Here we show another function for BAIBA, that of a bone-protective factor that prevents osteocyte cell death induced by reactive oxygen species (ROS). l-BAIBA was as or more protective than estrogen or N-acetyl cysteine, signaling through the Mas-Related G Protein-Coupled Receptor Type D (MRGPRD) to prevent the breakdown of mitochondria due to ROS. BAIBA supplied in drinking water prevented bone loss and loss of muscle function in the murine hindlimb unloading model, a model of osteocyte apoptosis. The protective effect of BAIBA was lost with age, not due to loss of the muscle capacity to produce BAIBA but likely to reduced Mrgprd expression with aging. This has implications for understanding the attenuated effect of exercise on bone with aging.

ACVR2B/Fc counteracts chemotherapy-induced loss of muscle and bone mass.

Chemotherapy promotes the development of cachexia, a debilitating condition characterized by muscle and fat loss. ACVR2B/Fc, an inhibitor of the Activin Receptor 2B signaling, has been shown to preserve muscle mass and prolong survival in tumor hosts, and to increase bone mass in models of osteogenesis imperfecta and muscular dystrophy. We compared the effects of ACVR2B/Fc on muscle and bone mass in mice exposed to Folfiri. In addition to impairing muscle mass and function, Folfiri had severe negative effects on bone, as shown by reduced trabecular bone volume fraction (BV/TV), thickness (Tb.Th), number (Tb.N), connectivity density (Conn.Dn), and by increased separation (Tb.Sp) in trabecular bone of the femur and vertebra. ACVR2B/Fc prevented the loss of muscle mass and strength, and the loss of trabecular bone in femurs and vertebrae following Folfiri administration. Neither Folfiri nor ACVR2B/Fc had effects on femoral cortical bone, as shown by unchanged cortical bone volume fraction (Ct.BV/TV), thickness (Ct.Th) and porosity. Our results suggest that Folfiri is responsible for concomitant muscle and bone degeneration, and that ACVR2B/Fc prevents these derangements. Future studies are required to determine if the same protective effects are observed in combination with other anticancer regimens or in the presence of cancer.

Smad2 overexpression induces alveolar bone loss and up regulates TNF-α, and RANKL.

OBJECTIVE: The aim of the current study was to investigate whether Smad2 overexpression in JE cells induced alveolar bone loss, and to understand the mechanisms regulating the bone loss. METHODS: A mouse line was created that used a cytokeratin 14 (K14) promoter to overexpress Smad2 in the epithelium of the transgenic mice (K14-Smad2). Micro CT radiographs (µCT) were used to assess bone loss, bone volume, and bone density. The expression of Tnfα, Il1-ß, Ifγ, Rankl, and Opg were assessed by RT-PCR. Western blots were used to detect the protein levels of TNF-α and IL1-ß. Tartrate-resistant acid phosphatase (TRAP) was used as a marker for osteoclasts. Wild type (WT) mice were used as controls in all steps of the current study. RESULTS: K14-Smad2 mice had 52.5% (±4.2) root exposed compared to 32.4%(±3.2) in the WT mice. There was a significant difference in alveolar bone volume in the K14-Smad2 mice when compared to WT mice 2.65mm3 (±0.3) and 4.3mm3 (±0.35) respectively. K14-Smad2 mice also had reduced bone density 696.8mg/cc (±70) at 12 months when compared to WT mice 845.9mg/cc(±10). The mRNA levels of Tnfα and Rankl increased by 3.26- and 2.5-fold respectively in the K14-Smad2 mice when compared to controls. The protein level of TNF-α was also significantly increased to 2.8-fold in K14-Smad2 mice when compared to WT mice. Smad2 overexpression increased the total numbers of osteoclasts in K14-Smad2 mice (3.4±0.2)-fold when compared to WT mice. CONCLUSION: Smad2 overexpression induces alveolar bone loss and increases the numbers of osteoclasts. Also, Smad2 overexpression up-regulates TNF-α and RANKL.

Microtubule disassembly prevents palatal fusion and alters regulation of the E-cadherin/catenin complex.

During palatal fusion, the midline epithelial seam (MES) degrades to achieve mesenchymal confluence. Epithelial mesenchymal transition (EMT) is one mechanism which is active in MES degradation. TGF-ß induces EMT in medial edge epithelium (MEE) by down-regulation of an epithelial marker, E-cadherin. Microtubule disassembly impaired palatal fusion leading to a multi-layered MES in the mid-region. In this study, we investigated the effect of microtubule disruption on the regulation of the E-cadherin/catenin adhesion complex. Nocodazole (NDZ) enhanced the accumulation of the adhesion complex at cell-cell contacts in MEE, while loss of the adhesion complex was observed in the control. NDZ caused aberrant regulation of the E-cadherin transcriptional repressors (Snail and Zeb) and the activator (c-MYC) through inhibition of the TGF-ß/SMAD2 signaling pathway, which led to a failure in EMT. These results suggest that the microtubule cytoskeleton plays an important role in mediating TGF-ß/SMAD2 signals to control E-cadherin gene expression in MEE during palatal fusion.

Smad2 is involved in the apoptosis of murine gingival junctional epithelium associated with inhibition of Bcl-2.

OBJECTIVE: Gingival junctional epithelium (JE) actively contributes to the homeostasis of the periodontium. Altered activation of TGF-ß signalling is implicated in the epithelium from chronic periodontitis. However, little is known about the effects of TGF-ß signalling on the JE. In this study, we investigated the relationship between Smad2, which plays an important role in mediating TGF-ß signal, and induction of apoptosis in the JE. METHODS: K14-Smad2 transgenic mice were used to observe the effect of over-expression of Smad2 driven by CK14 promoter in the JE. We performed TUNEL technique to evaluate the epithelial apoptosis. Expression of apoptosis related genes was examined using real-time PCR and immunofluorescence. RESULTS: K14-Smad2 mice showed an increased number of phospho-Smad2 positive JE cells associated with an increase in TGF-ß1 expression. K14-Smad2 mice have a significantly higher percentage of TUNEL positive cells in the JE. Immunofluorescence double labelling revealed that TUNEL positive cells showed immunoreactivity to phospho-Smad2. Real-time PCR analysis of apoptosis related gene expression provided evidence of lower expression of Bcl-2 in the gingival tissue from K14-Smad2 mice. There was a strong positive reaction for Bcl-2 protein in the junctional epithelium of wild type mice, while the gingival tissue of K14-Smad2 transgenic mice had only a faint signal for Bcl-2. CONCLUSIONS: The present study provided evidence that Smad2 plays a crucial role in the induction of apoptosis in gingival JE through inhibition of Bcl-2.

Multi-layered hypertrophied MEE formation by microtubule disruption via GEF-H1/RhoA/ROCK signaling pathway.

BACKGROUND: Formation of the secondary palate is complex and disturbance during palatal fusion may result in cleft palate. The processes of adhesion, intercalation, and disappearance of medial edge epithelia (MEE) are characterized by morphological changes requiring dynamic cytoskeletal rearrangement. Microtubules are one of the cytoskeletal elements involved in maintenance of cell morphology. Microtubule-disrupting drugs have been reported to cause craniofacial malformations including cleft palate. The mechanisms underlying the failure of palatal fusion remain poorly understood. We evaluated the effect of nocodazole (NDZ), a drug that disrupts microtubules, on palatal fusion in organ culture. RESULTS: NDZ caused failure of palatal fusion due to the induction of a multi-layered hypertrophied MEE in the mid-region of the secondary palatal shelves. Microtubule disruption increased RhoA activity and stress fiber formation. Pharmacological inhibition of the RhoA/ROCK pathway blocked multi-layered MEE formation. Partial prevention of hypertrophied MEE was observed with Y27632 and cytochalasin, but not with blebbistatin. NDZ induced re-localization of GEF-H1 into cytoplasm from cell-cell junctions. CONCLUSIONS: The present study provided evidence that the GEF-H1/RhoA/ROCK pathway plays a pivotal role in linking microtubule disassembly to the remodeling of the actin cytoskeleton, which resulted in a multi-layered hypertrophied MEE and failure of palatal fusion.

Mechanical induction of PGE2 in osteocytes blocks glucocorticoid-induced apoptosis through both the ß-catenin and PKA pathways.

Glucocorticoids are known to induce osteocyte apoptosis, whereas mechanical loading has been shown to sustain osteocyte viability. Here we show that mechanical loading in the form of fluid-flow shear stress blocks dexamethasone-induced apoptosis of osteocyte-like cells (MLO-Y4). Prostaglandin E(2) (PGE(2) ), a rapidly induced signaling molecule produced by osteocytes, was shown to be protective against dexamethasone-induced apoptosis, whereas indomethacin reversed the antiapoptotic effects of shear stress. This protective effect of shear stress was mediated through EP2 and EP4 receptors, leading to activation of the cAMP/protein kinase A signaling pathway. Activation of phosphatidylinositol 3-kinase, an inhibitor of glycogen synthesis kinase 3, also occurred, leading to the nuclear translocation of ß-catenin, an important signal transducer of the Wnt signaling pathway. Both shear stress and prostaglandin increased the phosphorylation of glycogen synthesis kinase 3 α/ß. Lithium chloride, an activator of the Wnt pathway, also was protective against glucocorticoid-induced apoptosis. Whereas it is known that mechanical loading increases cyclooxygenase-2 and EP2 receptor expression and prostaglandin production, dexamethasone was shown to inhibit expression of these components of the prostaglandin pathway and to reduce ß-catenin protein expression. ß-catenin siRNA knockdown experiments abrogated the protective effects of PGE(2), confirming the central role of ß-catenin in mediating the protection against dexamethasone-induced cell death. Our data support a central role for PGE(2) acting through the cAMP/PKA and ß-catenin signaling pathways in the protection of osteocyte apoptosis by fluid-flow shear stress.