Ming H. Zheng

Gene Expression of Osteoprotegerin Ligand, Osteoprotegerin, and Receptor Activator of NF-κB in Giant Cell Tumor of Bone: Possible Involvement in Tumor Cell-Induced Osteoclast-Like Cell Formation

Giant cell tumor of bone (GCT) is a rare primary osteolytic tumor of bone that is characterized by massive tissue destruction at the epiphysis of long bones. There is no evidence that tumor cells themselves are capable of bone destruction; instead, it appears that the tumor cells of GCT act by promoting osteoclastogenesis and, as a consequence, osteoclastic bone resorption. However, the mechanism by which this is achieved is not understood. Here we attempted to determine whether osteoprotegerin ligand (OPGL), the factor that is necessary and essential for osteoclastogenesis, is involved in tumor cell-recruited osteoclast-like giant cell formation in GCT. Using fluorescence in situ hybridization, we sought to determine mRNA expression of OPGL, its receptor RANK, and its decoy receptor OPG in three major cell types of GCT. We demonstrated that OPG mRNA was expressed in all three cell types of GCT, OPGL transcripts were mainly detected in spindle-shaped stromal-like tumor cells, whereas RANK was expressed only in macrophage-like mononuclear cells and multinuclear osteoclast-like giant cells. By semiquantitative RT-PCR, we also showed that the level of OPGL mRNA in GCT is much higher than that in normal bone and osteogenic osteosarcoma. In contrast, a similar level of OPG transcripts was detected in these three kinds of tissues, and RANK mRNA was detectable only in GCT tissues. We have further examined the regulation of gene expression of OPGL and OPG in tumor cells in response to osteotropic hormones. Administration of 1,25(OH)(2)D(3) and dexamethasone resulted in maximum up-regulation of OPGL level and down-regulation of OPG level in cultured GCT stromal-like tumor cells and the mouse bone marrow-derived ST-2 stromal cell line. Furthermore, we have shown that tumor cells of GCT induce differentiation of RANK-expressing myeloid RAW(264.7) cells into osteoclast-like cells in the presence of 1,25(OH)(2)D(3) and dexamethasone. Our findings suggest that OPGL is involved in the tumor cell-induced osteoclast-like cell formation in GCT. The ratio of OPGL/OPG by tumor cells may contribute to the degree of osteoclastogenesis and bone resorption.

Gene expression profiles of human chondrocytes during passaged monolayer cultivation

Chondrocyte phenotype has been shown to dedifferentiate during passaged monolayer cultivation. Hence, we have investigated the expression profile of 27 chondrocyte‐associated genes from both osteoarthritic cartilage tissue and healthy passaged human articular chondrocytes by quantitative real‐time PCR. Our results indicate that the gene expression levels of matrix proteins and proteases in chondrocytes from monolayer culture decrease compared with those from cartilage tissue, while monolayer cultured chondrocytes from normal and osteoarthritic cartilage exhibit similar gene expression patterns. However, chondrocytic gene expression profiles were differentially altered at various stages of passage. The expression of the matrix proteins aggrecan, type II collagen, and fibromodulin inversely correlated with increasing passage number, while fibronectin and link protein exhibited a marked increase with passage. The expression of matrix proteinases MMP‐3/9/13 and ADAMTS‐4/5 decreased with passage, whereas proteinase inhibitors TIMP‐2/3 were elevated. The cytokine IL‐1 also showed increased expression with monolayer chondrocyte culture, while IGF‐1 expression levels were diminished. No significant changes in TGF‐β, or the chondrogenic transcription factors Sox‐9, c‐fos, or c‐jun were observed. Our data indicates that cultured chondrocytes undergo dedifferentiation during monolayer culture, although the gene expression level of transcription factors necessary for chondrogenesis remains unchanged. This data may prove important for the future development of more specific and efficacious cultivation techniques for human articular chondrocyte‐based therapies.

Subchondral bone in osteoarthritis: insight into risk factors and microstructural changes

Osteoarthritis (OA) is a major cause of disability in the adult population. As a progressive degenerative joint disorder, OA is characterized by cartilage damage, changes in the subchondral bone, osteophyte formation, muscle weakness, and inflammation of the synovium tissue and tendon. Although OA has long been viewed as a primary disorder of articular cartilage, subchondral bone is attracting increasing attention. It is commonly reported to play a vital role in the pathogenesis of OA. Subchondral bone sclerosis, together with progressive cartilage degradation, is widely considered as a hallmark of OA. Despite the increase in bone volume fraction, subchondral bone is hypomineralized, due to abnormal bone remodeling. Some histopathological changes in the subchondral bone have also been detected, including microdamage, bone marrow edema-like lesions and bone cysts. This review summarizes basic features of the osteochondral junction, which comprises subchondral bone and articular cartilage. Importantly, we discuss risk factors influencing subchondral bone integrity. We also focus on the microarchitectural and histopathological changes of subchondral bone in OA, and provide an overview of their potential contribution to the progression of OA. A hypothetical model for the pathogenesis of OA is proposed.

Cloning, Sequencing, and Functional Characterization of the Rat Homologue of Receptor Activator of NF-κB Ligand†

A complementary DNA (cDNA) encoding the rat homologue of receptor activator of NF‐κB ligand/osteoprotegerin ligand/osteoclast differentiation factor/tumor necrosis factor (TNF)‐related activation‐induced cytokine (RANKL/OPGL/ODF/TRANCE) was cloned and sequenced from tibias of ovariectomized (OVX) rats. The predicted amino acid sequence of rat RANKL (rRANKL) has 84% and 96% identity to that of human and mouse RANKL, respectively, and 35% and 37% similarity to that of human and mouse TNF‐related apoptosis‐inducing ligand (TRAIL), respectively. RANKL transcripts were expressed abundantly in the thymus and bone tissues of OVX rats. rRANKL has a single hydrophobic region between residues 53 and 69, which is most likely to serve as a transmembrane domain. The long C‐terminal region containing β‐sheet‐forming sequences of the TNF‐like core is considered the extracellular region. Three truncated domains within the TNF‐like core region were expressed as glutathione S‐transferase (GST) fusion proteins and investigated for their ability to induce osteoclastogenesis. The results showed that GST‐rRANKL (aa160‐318) containing the full TNF‐like core region had the highest capability to induce the formation of osteoclast‐like cells from RAW264.7 cells. GST‐rRANKL (aa239‐318 and aa160‐268) had lesser degrees of osteoclast inductivity. Furthermore, the GST‐rRANKL (aa160‐318) is capable of (1) inducing osteoclast formation from rat spleen cells in the presence of macrophage colony‐stimulating factor (M‐CSF), (2) stimulating mature rat osteoclast polarization and bone resorption ex vivo, and (3) inducing systemic hypercalcemia in vivo; thus the full TNF‐like core region of rRANKL is an important regulator of calcium homeostasis and osteoclastic function.

NF-κB modulators in osteolytic bone diseases

Osteoclasts are responsible for bone resorption and play a pivotal role in the pathogenesis of osteolytic disorders. NF-kappaB is a set of nuclear factors that bind to consensus DNA sequences called kappaB sites, and is essential for osteoclast formation and survival. NF-kappaB signalling pathways are strictly regulated to maintain bone homeostasis by cytokines such as RANKL, TNF-alpha and IL-1, which differentially regulate classical and/or alternative NF-kappaB pathways in osteoclastic cells. These pathways are also modulated by NF-kappaB mediators, including TRAF6, aPKC, p62/SQSTM1 and deubiquitinating enzyme CYLD that are involved in the ubiquitin-proteasome system during RANK-mediated osteoclastogenesis. Abnormal activation of NF-kappaB signalling in osteoclasts has been associated with excessive osteoclastic activity, and frequently observed in osteolytic conditions, including periprosthetic osteolysis, arthritis, Pagets disease of bone, and periodontitis. NF-kappaB modulators such as parthenolide and NEMO-binding domain peptide demonstrate therapeutic effects on inflammation-induced bone destruction in mouse models. Unravelling the structure and function of NF-kappaB pathways in osteoclasts and other cell types will be important in developing new strategies for treatments of bone diseases.

12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits osteoclastogenesis by suppressing RANKL-induced NF-κB activation

The mechanism by which TPA‐induced PKC activity modulates osteoclastogenesis is not clear. Using a RAW264.7 cell culture system and assays for NF‐κB nuclear translocation, NF‐κB reporter gene activity, and MAPK assays, we demonstrated that TPA inhibits osteoclastogenesis through the suppression of RANKL‐induced NF‐κβ activation.

Effects of Bafilomycin A1: an inhibitor of vacuolar H (+)-ATPases on endocytosis and apoptosis in RAW cells and RAW cell-derived osteoclasts.

Bafilomycin A1, a specific inhibitor of V‐ATPases, is a potent inhibitor of bone resorption, but the underlying mechanisms of its action remain unclear. In this study, we investigated the effect of Bafilomycin A1 on endocytosis and apoptosis in RAW cells and RAW cell‐derived osteoclasts. Quantitative analysis by flow cytometry showed that Bafilomycin A1 increased total transferrin levels when RAW cells were exposed to labeled transferrin and decreased the total uptake of Dextran‐rhodamine B, both in a dose‐ and time‐dependent fashion, indicating that Bafilomycin influences receptor‐mediated and fluid phase endocytosis in these cells. Furthermore, Bafilomycin A1 induced apoptosis of RAW cells in a dose dependent manner as evidenced by Annexin V flow cytometry. The action of Bafilomycin A1 on endocytotic events appeared to be more sensitive and occurred earlier than on its apoptosis inducing effects, suggesting that interrupting of endocytosis might be an early sign of Bafilomycin‐mediated osteoclast inhibition. Semi‐quantitative RT‐PCR analysis showed that the gene transcripts of putative Bafilomycin A1 binding subunit, V‐ATPase‐subunit a3, were expressed in the preosteoclastic RAW cell line, and up‐regulated during RANKL‐induced osteoclastogenesis. Osteoclasts treated with Bafilomycin A1 exhibited apoptosis as well as altered cellular localization of Transferrin Alexa 647. Given that endocytosis and apoptosis are important processes during osteoclastic bone resorption, the potent effect of Bafilomycin A1 on endocytosis and apoptosis of osteoclasts and their precursor cells may in part account for Bafilomycin A1 inhibited bone resorption. J. Cell. Biochem. 88: 1256–1264, 2003.

Sesquiterpene Lactone Parthenolide Blocks Lipopolysaccharide‐Induced Osteolysis Through the Suppression of NF‐κB Activity

Effective treatment for bacteria‐induced bone lytic diseases is not yet available. In this study, we showed that PAR, an NF‐κB inhibitor found in medicinal herbs, can block LPS‐induced osteolysis. PAR does this by inhibiting osteoclastogenesis and bone resorption and promoting apoptosis of osteoclasts through the suppression of NF‐κB activity.

Naringin abrogates osteoclastogenesis and bone resorption via the inhibition of RANKL‐induced NF‐κB and ERK activation

Osteolytic bone diseases including osteoporosis are commonly accompanied with enhanced osteoclast formation and bone resorption. Naringin, a natural occurring flavonoid has been found to protect against retinoic acid‐induced osteoporosis and improve bone quality in rats. Here, we showed that naringin perturbs osteoclast formation and bone resorption by inhibiting RANK‐mediated NF‐κB and ERK signaling. Naringin suppressed gene expression of key osteoclast marker genes. Naringin was found to inhibit RANKL‐induced activation of NF‐κB by suppressing RANKL‐mediated IκB‐α degradation. In addition, naringin inhibited RANKL‐induced phosphorylation of ERK. This study identifies naringin as an inhibitor for osteoclast formation and bone resorption, and provides evidence that natural compounds such as naringin might be beneficial as an alternative medicine for the prevention and treatment of osteolysis.

Calcium/calmodulin-dependent kinase activity is required for efficient induction of osteoclast differentiation and bone resorption by receptor activator of nuclear factor kappa B ligand (RANKL).

Calcium/calmodulin‐dependent protein kinase (CaMK) is a major down stream mediator of Ca2+ signaling in a wide range of cellular functions, including ion channel and cell cycle regulation and neurotransmitter synthesis and release. Here we have investigated the role of the CaMK signaling pathway in osteoclast differentiation and bone resorption. We observed that the CaMKI, CaMKII γ isoforms were present in both bone‐marrow derived macrophages and RAW264.7 murine macrophage cell line, and that expression persisted during osteoclast differentiation in the presence of receptor activator of nuclear factor kappa B (NF‐κB) ligand (RANKL). RANKL‐induced differentiation was accompanied by increased cyclic AMP response element transcriptional activity, and ERK phosphorylation, which are both downstream targets of CaMK. Two selective inhibitors of CaMKs, KN‐93 and KN‐62, inhibited osteoclastogenesis in a time and concentration‐dependent manner. This was accompanied by suppression of cathepsin K expression and osteoclastic bone resorption, which are markers for differentiated osteoclast function. KN‐93 and KN‐62 both inhibited RANKL‐induced ERK phosphorylation and CREB transcriptional activity. These findings imply a role for CaMK in osteoclast differentiation and bone resorption. J. Cell. Physiol. 212:787–795, 2007.