Shek Man Chim

An overview of the regulation of bone remodelling at the cellular level.

OBJECTIVES To review the current literature on the regulation of bone remodelling at the cellular level. DESIGN AND METHODS The cellular activities of the cells in the basic multicellular unit (BMU) were evaluated. RESULTS Bone remodelling requires an intimate cross-talk between osteoclasts and osteoblasts and is tightly coordinated by regulatory proteins that interact through complex autocrine/paracrine mechanisms. Osteocytes, bone lining cells, osteomacs, and vascular endothelial cells also regulate bone remodelling in the BMU via cell signalling networks of ligand-receptor complexes. In addition, through secreted and membrane-bound factors in the bone microenvironment, T and B lymphocytes mediate bone homeostasis in osteoimmunology. CONCLUSIONS Osteoporosis and other bone diseases occur because multicellular communication within the BMU is disrupted. Understanding the cellular and molecular basis of bone remodelling and the discovery of novel paracrine or coupling factors, such as RANKL, sclerostin, EGFL6 and semaphorin 4D, will lay the foundation for drug development against bone diseases.

Angiogenic factors in bone local environment.

Angiogenesis plays an important role in physiological bone growth and remodeling, as well as in pathological bone disorders such as fracture repair, osteonecrosis, and tumor metastasis to bone. Vascularization is required for bone remodeling along the endosteal surface of trabecular bone or Haversian canals within the cortical bone, as well as the homeostasis of the cartilage-subchondral bone interface. Angiogenic factors, produced by cells from a basic multicellular unit (BMU) within the bone remodeling compartment (BRC) regulate local endothelial cells and pericytes. In this review, we discuss the expression and function of angiogenic factors produced by osteoclasts, osteoblasts and osteocytes in the BMU and in the cartilage-subchondral bone interface. These include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), BMP7, receptor activator of NF-κB ligand (RANKL) and epidermal growth factor (EGF)-like family members. In addition, the expression of EGFL2, EGFL3, EGFL5, EGFL6, EGFL7, EGFL8 and EGFL9 has been recently identified in the bone local environment, giving important clues to their possible roles in angiogenesis. Understanding the role of angiogenic factors in the bone microenvironment may help to develop novel therapeutic targets and diagnostic biomarkers for bone and joint diseases, such as osteoporosis, osteonecrosis, osteoarthritis, and delayed fracture healing.

Prevention of Wear Particle-Induced Osteolysis by a Novel V-ATPase Inhibitor Saliphenylhalamide through Inhibition of Osteoclast Bone Resorption

Wear particle-induced peri-implant loosening (Aseptic prosthetic loosening) is one of the most common causes of total joint arthroplasty. It is well established that extensive bone destruction (osteolysis) by osteoclasts is responsible for wear particle-induced peri-implant loosening. Thus, inhibition of osteoclastic bone resorption should prevent wear particle induced osteolysis and may serve as a potential therapeutic avenue for prosthetic loosening. Here, we demonstrate for the first time that saliphenylhalamide, a new V-ATPase inhibitor attenuates wear particle-induced osteolysis in a mouse calvarial model. In vitro biochemical and morphological assays revealed that the inhibition of osteolysis is partially attributed to a disruption in osteoclast acidification and polarization, both a prerequisite for osteoclast bone resorption. Interestingly, the V-ATPase inhibitor also impaired osteoclast differentiation via the inhibition of RANKL-induced NF-κB and ERK signaling pathways. In conclusion, we showed that saliphenylhalamide affected multiple physiological processes including osteoclast differentiation, acidification and polarization, leading to inhibition of osteoclast bone resorption in vitro and wear particle-induced osteolysis in vivo. The results of the study provide proof that the new generation V-ATPase inhibitors, such as saliphenylhalamide, are potential anti-resorptive agents for treatment of peri-implant osteolysis.

EGFL6 Promotes Endothelial Cell Migration and Angiogenesis through the Activation of Extracellular Signal-regulated Kinase

Angiogenesis is required for bone development, growth, and repair. It is influenced by the local bone environment that involves cross-talks between endothelial cells and adjacent bone cells. However, data regarding factors that directly contribute to angiogenesis by bone cells remain poorly understood. Here, we report that EGFL6, a member of the epidermal growth factor (EGF) repeat superfamily proteins, induces angiogenesis by a paracrine mechanism in which EGFL6 is expressed in osteoblastic-like cells but promotes migration and angiogenesis of endothelial cells. Co-immunoprecipitation assays revealed that EGFL6 is secreted in culture medium as a homodimer protein. Using scratch wound healing and transwell assays, we found that conditioned medium containing EGFL6 potentiates SVEC (a simian virus 40-transformed mouse microvascular endothelial cell line) endothelial cell migration. In addition, EGFL6 promotes the endothelial cell tube-like structure formation in Matrigel assays and angiogenesis in a chick embryo chorioallantoic membrane. Furthermore, we show that EGFL6 recombinant protein induces phosphorylation of ERK in SVEC endothelial cells. Inhibition of ERK impaired EGFL6-induced ERK activation and endothelial cell migration. Together, these results demonstrate, for the first time, that osteoblastic-like cells express EGFL6 that is capable of promoting endothelial cell migration and angiogenesis via ERK activation. Thus, the EGLF6 mediates a paracrine mechanism of cross-talk between vascular endothelial cells and osteoblasts and might offer an important new target for the potential treatment of bone diseases, including osteonecrosis, osteoporosis, and fracture healing.

miR‐764‐5p promotes osteoblast differentiation through inhibition of CHIP/STUB1 expression

Differentiation of committed precursor cells into the osteoblast lineage is tightly regulated by several factors, including Runx2 and BMP2. We previously reported that C terminus of Hsc70‐interacting protein/STIP1 homology and U‐Box containing protein 1 (CHIP/STUB1) negatively regulated osteoblast differentiation through promoting Runx2 protein degradation. However, how CHIP is regulated during osteoblast differentiation remains unknown. In this study, we found that miR‐764‐5p is up‐expressed during the osteoblast differentiation in calvarial and osteoblast progenitor cells, coupled with down‐expression of CHIP protein. We observed that forced expression or inhibition of miR‐764‐5p decreased or increased the CHIP protein level through affecting its translation by targeting the 3′‐UTR region. Perturbation of miR‐764‐5p resulted in altered differentiation fate of osteoblast progenitor cells and the role of miR‐764‐5p was reversed by overexpression of CHIP, whereas depletion of CHIP impaired the effect of miR‐764‐5p. Our data showed that miR‐764‐5p positively regulates osteoblast differentiation from osteoblast progenitor cells by repressing the translation of CHIP protein.

Microstructural analysis of collagen and elastin fibres in the kangaroo articular cartilage reveals a structural divergence depending on its local mechanical environment

OBJECTIVE To assess the microstructure of the collagen and elastin fibres in articular cartilage under different natural mechanical loading conditions and determine the relationship between the microstructure of collagen and its mechanical environment. METHOD Articular cartilage specimens were collected from the load bearing regions of the medial femoral condyle and the medial distal humerus of adult kangaroos. The microstructure of collagen and elastin fibres of these specimens was studied using laser scanning confocal microscopy (LSCM) and the orientation and texture features of the collagen were analysed using ImageJ. RESULTS A zonal arrangement of collagen was found in kangaroo articular cartilage: the collagen fibres aligned parallel to the surface in the superficial zone and ran perpendicular in the deep zone. Compared with the distal humerus, the collagen in the femoral condyle was less isotropic and more clearly oriented, especially in the superficial and deep zones. The collagen in the femoral condyle was highly heterogeneous, less linear and more complex. Elastin fibres were found mainly in the superficial zone of the articular cartilage of both femoral condyle and distal humerus. CONCLUSIONS The present study demonstrates that the collagen structure and texture of kangaroo articular cartilage is joint-dependent. This finding emphasizes the effects of loading on collagen development and suggests that articular cartilage with high biochemical and biomechanical qualities could be achieved by optimizing joint loading, which may benefit cartilage tissue engineering and prevention of joint injury. The existence of elastin fibres in articular cartilage could have important functional implications.

SC-514, a selective inhibitor of IKKβ attenuates RANKL-induced osteoclastogenesis and NF-κB activation

The RANKL-induced NF-κB signaling pathway is essential for osteoclastogenesis. This study aims to identify specific inhibitors targeting NF-κB signaling pathway, which might serve as useful small molecule inhibitors for the treatment and alleviation of osteoclast-mediated bone lytic diseases. By screening for compounds that selectively inhibit RANKL-induced NF-κB activation in RAW264.7 cells as monitored by luciferase reporter gene assay, we identified SC-514, a specific inhibitor of IKKβ, as a candidate compound targeting osteoclastogenesis. SC-514 dose-dependently inhibits RANKL-induced osteoclastogenesis with an IC50 of <5μM. At high concentrations, SC-514 (≥12.5μM) induced apoptosis and caspase 3 activation in RAW264.7 cells. Moreover, SC-514 specifically suppressed NF-κB activity owing to delayed RANKL-induced degradation of IκBα and inhibition of p65 nuclear translocation. Taken together, our results indicate that SC-514 impairs RANKL-induced osteoclastogenesis and NF-κB activation. Thus, targeting IKKβ by SC-514 presents as a potential treatment for osteoclast-related disorders such as osteoporosis and cancer-induced bone loss.

HtrA1 is upregulated during RANKL‐induced osteoclastogenesis, and negatively regulates osteoblast differentiation and BMP2‐induced Smad1/5/8, ERK and p38 phosphorylation

Bone remodeling is regulated by secreted factors in the bone microenvironment. However, data regarding osteoclast‐derived factors that influence osteoblast differentiation are lacking. Here, we show that HtrA1 is produced as a secreted protein during osteoclastogenesis, and negatively regulates osteoblast differentiation. Exogenous addition of recombinant HtrA1 attenuates osteoblast differentiation and BMP2‐induced Smad1/5/8, ERK1/2 and p38 phosphorylation in pre‐osteoblasts. Our studies imply a unique mode of crosstalk in which HtrA1 is produced by both osteoclasts and osteoblasts and negatively regulates osteoblast differentiation, suggesting that HtrA1 may mediate the fine tuning of paracrine and autocrine regulations during bone remodeling processes.

Triptolide inhibits osteoclast formation, bone resorption, RANKL-mediated NF-қB activation and titanium particle-induced osteolysis in a mouse model.

The RANKL-induced NF-κB signaling pathway is required for osteoclast formation and function. By screening for compounds that inhibit RANKL-induced NF-κB activation using a luciferase reporter gene assay in RAW264.7 cells, we identified triptolide (PG490), as a candidate compound targeting osteoclast differentiation and osteoclast-mediated osteolysis. Triptolide (PG490) is an active compound of the medicinal herb Tripterygium wilfordii Hook F (TWHF) or Lei Gong Teng with known anti-inflammatory properties. We found that triptolide inhibited osteoclastogenesis and bone resorption, as well as RANKL-induced NF-қB activities as monitored by luciferase reporter gene assays and the nuclear translocation of p65. In vivo studies showed that triptolide attenuates titanium-induced osteolysis and osteoclast formation in a mouse calvarial model. Considering that drugs which protect against localized bone loss are critically needed for the effective treatment of particle-induced osteolysis, our data suggest that triptolide might have therapeutic potential for the treatment of bone lytic diseases caused by prosthetic wear particles.

EGFL7 is expressed in bone microenvironment and promotes angiogenesis via ERK, STAT3, and integrin signaling cascades.

Angiogenesis plays a pivotal role in bone formation, remodeling, and fracture healing. The regulation of angiogenesis in the bone microenvironment is highly complex and orchestrated by intercellular communication between bone cells and endothelial cells. Here, we report that EGF‐like domain 7 (EGFL7), a member of the epidermal growth factor (EGF) repeat protein superfamily is expressed in both the osteoclast and osteoblast lineages, and promotes endothelial cell activities. Addition of exogenous recombinant EGFL7 potentiates SVEC (simian virus 40‐transformed mouse microvascular endothelial cell line) cell migration and tube‐like structure formation in vitro. Moreover, recombinant EGFL7 promotes angiogenesis featuring web‐like structures in ex vivo fetal mouse metatarsal angiogenesis assay. We show that recombinant EGFL7 induces phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2), signal transducer and activator of transcription 3 (STAT3), and focal adhesion kinase (FAK) in SVEC cells. Inhibition of ERK1/2 and STAT3 signaling impairs EGFL7‐induced endothelial cell migration, and angiogenesis in fetal mouse metatarsal explants. Bioinformatic analyses indicate that EGFL7 contains a conserved RGD/QGD motif and EGFL7‐induced endothelial cell migration is significantly reduced in the presence of RGD peptides. Moreover, EGFL7 gene expression is significantly upregulated during growth plate injury repair. Together, these results demonstrate that EGFL7 expressed by bone cells regulates endothelial cell activities through integrin‐mediated signaling. This study highlights the important role that EGFL7, like EGFL6, expressed in bone microenvironment plays in the regulation of angiogenesis in bone. J. Cell. Physiol. 230: 82–94, 2015.