Dermatan sulfate inhibits osteoclast formation by binding to receptor activator of NF-kappa B ligand

Dermatan sulfate (DS) is a major component of extracellular matrices in mammalian tissues. In the present study, DS demonstrated a high level of binding activity to receptor activator of NF-kappaB ligand (RANKL) and obstructed the binding of RANK to RANKL, determined using a quartz-crystal microbalance (QCM) technique. Further, when mouse bone marrow cells were cultured with RANKL and macrophage colony-stimulating factor, DS suppressed tartrate-resistant acid phosphatase-positive multinucleated cell formation in a dose-dependent manner. In addition, immunoblot analyses revealed that DS reduced the levels of phosphorylation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase protein in mouse osteoclast progenitor cells stimulated with RANKL. Together, these results indicate that DS regulates osteoclast formation through binding to RANKL and inhibition of signal transduction in osteoclast progenitor cells, suggesting that it has an important role in bone metabolism in pathological conditions.     

Relevance of an in vitro osteoclastogenesis system to study receptor activator of NF-kB ligand and osteoprotegerin biological activities

Receptor activator of NF-kB Ligand (RANKL) is an essential requirement for osteoclastogenesis and its activity is neutralized by binding to the soluble decoy receptor osteoprotegerin (OPG). The purpose of this work was to study the effects of RANKL and OPG during osteoclastogenesis using the murine monocytic cell line RAW 264.7 that can differentiate into osteoclasts in vitro. RAW 264.7 cells plated at 10(4) cells/cm(2) and cultured for 4 days in the presence of RANKL represent the optimal culture conditions for osteoclast differentiation, with an up-regulation of all parameters related to bone resorption: tartrate resistant acid phosphatase (TRAP), calcitonin receptor (CTR), RANK, cathepsin K, matrix metalloproteinase (MMP)-9 mRNA expressions. RANKL and OPG biological effects vary according to the differentiation state of the cells: in undifferentiated RAW 264.7 cells, TRAP expression was decreased by OPG and RANKL, RANK expression was inhibited by OPG, while MMP-9 and cathepsin K mRNA expressions were not modulated. In differentiated RAW 264.7 cells, RANKL and OPG both exert an overall inhibitory effect on the expression of all the parameters studied. In these experimental conditions, OPG-induced MMP-9 inhibition was abrogated in the presence of a blocking anti-RANKL antibody, suggesting that part of OPG effects are RANKL-dependent.     

IL-6, leukemia inhibitory factor,and oncostatin M stimulate bone resorption and regulate the expression of receptor activator of NF-kappa B ligand,osteoprotegerin, and receptor activator of NF-kappa B in mouse calvariae

IL-6, leukemia inhibitory factor (LIF), and oncostatin M (OSM) are IL-6-type cytokines that stimulate osteoclast formation and function. In the present study, the resorptive effects of these agents and their regulation of receptor activator of NF-kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG) were studied in neonatal mouse calvaria. When tested separately, neither human (h) IL-6 nor the human soluble IL-6R (shIL-6R) stimulated bone resorption, but when hIL-6 and the shIL-6R were combined, significant stimulation of both mineral and matrix release from bone explants was noted. Semiquantitative RT-PCR showed that hIL-6 plus shIL-6R enhanced the expression of RANKL and OPG in calvarial bones, but decreased RANK expression. Human LIF, hOSM, and mouse OSM (mOSM) also stimulated 45Ca release and enhanced the mRNA expression of RANKL and OPG in mouse calvaria, but had no effect on the expression of RANK. In agreement with the RT-PCR analyses, ELISA measurements showed that both hIL-6 plus shIL-6R and mOSM increased RANKL and OPG proteins. 1,25-Dihydroxyvitamin D3 (D3) also increased the RANKL protein level, but decreased the protein level of OPG. OPG inhibited 45Ca release stimulated by RANKL, hIL-6 plus shIL-6R, hLIF, hOSM, mOSM, and D3. An Ab neutralizing mouse gp130 inhibited 45Ca release induced by hIL-6 plus shIL-6R. These experiments demonstrated stimulation of calvarial bone resorption and regulation of mRNA and protein expression of RANKL and OPG by D3 and IL-6 family cytokines as well as regulation of RANK expression in preosteoclasts/osteoclasts of mouse calvaria by D3 and hIL-6 plus shIL-6R.     

Osteopontin as a positive regulator in the osteoclastogenesis of arthritis

We examined the role of osteopontin (OPN) in the osteoclastogenesis of arthritis using collagen-induced arthritis (CIA). Cells from arthritic joints of wild-type (OPN +/+) mice spontaneously developed bone-resorbing osteoclast-like cells (OCLs). The cultured cells showed an enhanced expression of receptor activator of nuclear factor kappaB ligand (RANKL) and a decreased expression of osteoprotegerin (OPG). The addition of OPG reduced the number of OCLs, indicating that the osteoclastogenesis depends on the RANK/RANKL/OPG system. The cells also produced OPN abundantly and anti-OPN neutralizing antibodies suppressed the development of OCLs. Moreover, the addition of OPN increased the expression of RANKL and augmented differentiation of OCLs from OPN-deficient (OPN -/-) cells. OPN, like the combination of 1alpha,25-dihydroxyvitamin D(3) and dexamethasone, also enhanced the RANKL expression and decreased OPG expression in a stromal cell line, ST2. These results suggest that OPN acts as a positive regulator in the osteoclastogenesis of arthritis through the RANK/RANKL/OPG system.     

Epigenetic changes in the expression of the maize A1 gene in Petunia hybrida: role of numbers of integrated gene copies and state of methylation.

Summary ThePetunia hybrida mutant RL01 is white flowering due to a genetic block in the anthocyanin pathway. The introduction of the maize Al cDNA under the control of the CaMV 35S RNA promoter leads to the production of pelargonidin derivatives, resulting in a brick red flower phenotype. Among the transgenic petunia plants the pigmentation of the petals exhibited different expression patterns which could be categorized into the red, the variegated, and the white phenotype. This system proved to be especially suitable for the investigation of gene expression by simply looking at the pigmentation pattern of the petals. The uniformity of floral pelargonidin pigmentation is inversely correlated with the number of integrated Al copies. Furthermore, a correlation was found between the methylation status of the 35S RNA promoter and the instability of the floral pelargonidin coloration. The status of promoter methylation controlling the expression of the Al gene seems to be influenced by the copy number and the chromosomal position of the transferred gene.     

Prolactin decreases the expression ratio of receptor activator of nuclear factor kappaB ligand/osteoprotegerin in human fetal osteoblast cells

Prolactin (PRL) enhanced bone remodeling leading to net bone loss in adult and net bone gain in young animals. Studies in PRL-exposed osteoblasts derived from adult humans revealed an increase in the expression ratio of receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG), thus supporting the previous finding of PRL-induced bone loss in adults. This study thus investigated the effects of PRL on the osteoblast functions and the RANKL/OPG ratio in human fetal osteoblast (hFOB) cells which strongly expressed PRL receptors. After 48h incubation, PRL increased osteocalcin expression, but had no effect on cell proliferation. However, the alkaline phosphatase activity was decreased in a dose-response manner within 24h. The effect of PRL on alkaline phosphatase was abolished by LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor. PRL also decreased the RANKL/OPG ratio by downregulating RANKL and upregulating OPG expression, implicating a reduction in the osteoblast signal for osteoclastic bone resorption. It could be concluded that, unlike the osteoblasts derived from adult humans, PRL-exposed hFOB cells exhibited indices suggestive of bone gain, which could explain the in vivo findings in young rats. The signal transduction of PRL in osteoblasts involved the PI3K pathway.     

Receptor activator of NF-kappa B ligand and osteoprotegerin regulate proinflammatory cytokine production in mice

Receptor activator of NF-kappaB ligand (RANKL) is a membrane-bound or soluble cytokine essential for osteoclast differentiation, whereas the decoy receptor osteoprotegerin (OPG) masks RANKL activity. In mouse serum, both soluble RANKL and OPG are detectable. We observed that mice injected with LPS showed significantly down-regulated serum RANKL levels, whereas serum OPG levels were up-regulated. However, the roles of RANKL and OPG in innate immunity remain obscure. We found that RANKL pretreatment suppressed production of proinflammatory cytokines in macrophages in response to stimulation by bacteria and their components. Furthermore, such RANKL-induced tolerance in macrophages was inhibited by GM-CSF treatment, which blocks RANKL signaling. RANKL-induced tolerance occurred in the absence of c-Fos, which is essential for osteoclast differentiation. In mice lacking OPG, LPS-induced production of proinflammatory cytokines was reduced, whereas in mice lacking RANKL, it was increased, and lethality following LPS injection was also elevated, suggesting that constitutive activities of RANKL suppress cytokine responsiveness to LPS in vivo. Strikingly, prior administration of RANKL protected mice from LPS-induced death. These data reveal prophylactic potential of RANKL in acute inflammatory diseases.     

Curcumin (diferuloylmethane) inhibits receptor activator of NF-kappa B ligand-induced NF-kappa B activation in osteoclast precursors and suppresses osteoclastogenesis

Numerous studies have indicated that inflammatory cytokines play a major role in osteoclastogenesis, leading to the bone resorption that is frequently associated with cancers and other diseases. Gene deletion studies have shown that receptor activator of NF-kappaB ligand (RANKL) is one of the critical mediators of osteoclastogenesis. How RANKL mediates osteoclastogenesis is not fully understood, but an agent that suppresses RANKL signaling has potential to inhibit osteoclastogenesis. In this report, we examine the ability of curcumin (diferuloylmethane), a pigment derived from turmeric, to suppress RANKL signaling and osteoclastogenesis in RAW 264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated NF-kappaB, and preexposure of the cells to curcumin completely suppressed RANKL-induced NF-kappaB activation. Curcumin inhibited the pathway leading from activation of IkappaBalpha kinase and IkappaBalpha phosphorylation to IkappaBalpha degradation. RANKL induced osteoclastogenesis in these monocytic cells, and curcumin inhibited both RANKL- and TNF-induced osteoclastogenesis and pit formation. Curcumin suppressed osteoclastogenesis maximally when added together with RANKL and minimally when it was added 2 days after RANKL. Whether curcumin inhibits RANKL-induced osteoclastogenesis through suppression of NF-kappaB was also confirmed independently, as RANKL failed to activate NF-kappaB in cells stably transfected with a dominant-negative form of IkappaBalpha and concurrently failed to induce osteoclastogenesis. Thus overall these results indicate that RANKL induces osteoclastogenesis through the activation of NF-kappaB, and treatment with curcumin inhibits both the NF-kappaB activation and osteoclastogenesis induced by RANKL.     

The increased expression of receptor activator of nuclear‐κB ligand (RANKL) of multiple myeloma bone marrow stromal cells is inhibited by the bisphosphonate ibandronate

The receptor activator of nuclear factor‐kappaB ligand (RANKL) and interleukin‐1beta are osteoclast activating factors which are abnormally expressed in bone marrow stromal cells and plasma cells of multiple myeloma patients. In this work we analyzed RANKL expression in human bone marrow mesenchymal stromal cells and the effect of the bisphosphonate ibandronate on RANKL expression after IL‐1beta activation of ERK pathway. Mesenchymal stromal cells were obtained from bone marrow iliac aspirates from multiple myeloma patients at stages II/III and non‐osteoporotics control donors; these cells were maintained under long‐term culture conditions. Cells were cultured in the presence or the absence of 5 ng/ml IL‐1beta and/or 5 µM ibandronate, during selected periods. mRNA for RANKL and protein levels were assayed by RT‐PCR and Western blot, respectively. Human bone marrow stromal cell line HS‐5 was used for assessing IL 1beta‐ and ibandronate‐ERK phosphorylation responses. Multiple myeloma mesenchymal stromal cells differentiate from control cells by increased basal RANKL expression. IL‐1beta up regulated RANKL expression showed dependent on activated MEK/ERK pathway. Finally, the bisphosphonate ibandronate, that hindered activation of the MEK/ERK pathway significantly inhibited both basal and IL‐1beta dependent RANKL expression by cells. Results indicate that RANKL expression involves the MEK/ERK pathway in multiple myeloma mesenchymal stromal cells, and that early obstruction of this path, such as that achieved with ibandronate, significantly deters RANKL protein expression. J. Cell. Biochem. 111: 130–137, 2010. © 2010 Wiley‐Liss, Inc.