Mechanical inhibition of RANKL expression is regulated by H-Ras-GTPase

Mechanical input is known to regulate bone remodeling, yet the molecular events involved in mechanical signal transduction are poorly understood. We here investigate proximal events leading to the ERK1/2 activation that is required for mechanical repression of RANKL (receptor activator of NF-kappaB ligand) expression, the factor that controls local recruitment of osteoclasts. Using primary murine bone stromal cells we show that dynamic mechanical strain via substrate deformation activates Ras-GTPase, in particular the H-Ras isoform. Pharmacological inhibition of H-Ras function prevents strain activation of H-Ras as well as the downstream mechanical repression of RANKL. Furthermore, small interfering RNA silencing of H-Ras, but not K-Ras, abrogates mechanical strain repression of RANKL. H-Ras-specific inhibition of mechanorepression of RANKL was also demonstrated in a murine pre-osteoblast cell line (CIMC-4). The requirement of cholesterol for H-Ras activation was probed; cholesterol depletion of rafts using methyl-betacyclodextrin prevented mechanical H-Ras activation. That the mechanical repression of RANKL requires activation of H-Ras, a specific isoform of Ras-GTP that is known to reside in the lipid raft microdomain, suggests that spatial arrangements are critical for generation of specific downstream events in response to mechanical signals. By partitioning signals this way, cells may be able to generate different downstream responses through seemingly similar signaling cascades.     

OPG/RANKL/RANK系统与骨破坏性疾病

近年来发现的OPG/RANKL/RANK系统在破骨细胞生成中起着至关重要的作用,是骨骼生理研究领域的重大进展。成骨细胞、骨髓基质细胞、激活的T淋巴细胞表达RANKL,与破骨细胞前体细胞或成熟破骨细胞表面上的RANK结合后,促进破骨细胞的分化及骨吸收活性。成骨细胞及骨髓基质细胞分泌表达OPG可与RANKL竞争性结合,从而阻断RANKL与RANK之间的相互作用。体内多种激素或因子通过影响骨髓微环境内的OPG/RANKL比率来调节骨代谢。此外,乳腺上皮细胞表达有RANK,孕期在性激素的诱导下可表达RANKL,OPG/RANKL/RANK系统在孕期乳腺发育以及母体向胎儿的钙转运过程中发挥重要作用。阻断RANKL/RANK通路有望给骨质疏松、类风湿关节炎及癌症骨转移等骨破坏性疾病的治疗开辟新的途径。进一步研究应了解OPG/RANKL/RANK系统与其它信号传导途径的关系,重视骨骼、免疫及内分泌系统之间的相互作用。目前,开发与OPG功能相似或促进其表达的合成药物有可能成为具有良好经济效益和社会效益的产业。     

Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-kappaB ligand: modulation of the expression by osteotropic factors and cytokines

A variety of humoral factors modulate the osteoclastogenesis. Receptor activator of NF-kappaB ligand (RANKL) expressed on osteoblast/stromal lineage cells plays a pivotal role to transduce an essential differentiation signal to osteoclast lineage cells through binding to its receptor, RANK, expressed on the latter cell population; however, the difficulty to detect RANKL protein expression hampers us in investigating the regulation of RANKL expression by humoral factors. To determine protein expression of RANKL, we have established a new method, named as a ligand-receptor precipitation (LRP) Western blot analysis, which can specifically concentrate the target protein by the use of specific binding characteristic between RANKL and RANK/osteoprotegrin (OPG). RANKL protein expression in the postnuclear supernatant was not detected by common Western blotting, but LRP Western blot analysis clearly showed that RANKL is produced as a membrane-bound protein on murine osteoblasts/stromal cells, and cleaved into a soluble form by metalloprotease. Cytokines stimulating the osteoclastogenesis, such as IL-1beta, IL-6, IL-11, IL-17, and TNF-alpha, increased the expression of RANKL with decrease of OPG expression in osteoblasts/stromal cells. In contrast, cytokines inhibiting the osteoclastogenesis, such as IL-13, INF-gamma, and TGF-beta1 suppressed the expression of RANKL and/or augmented OPG expression. Functional difference between membrane-bound and soluble RANKL was demonstrated, which showed that membrane-bound RANKL works more efficiently than soluble RANKL in the osteoclastogenesis developed from murine bone marrow cell culture. The present study indicates the usefulness of LRP Western blot analysis, which shows that the modulation of osteoclastogenesis by humoral factors is achieved, in part, by regulation of the expression of RANKL and OPG in osteoblast/stromal lineage cells.     

Inflammatory T cells rapidly induce differentiation of human bone marrow stromal cells into mature osteoblasts

Activated T cells secrete multiple osteoclastogenic cytokines which play a major role in the bone destruction associated with rheumatoid arthritis. While the role of T cells in osteoclastogenesis has received much attention recently, the effect of T cells on osteoblast formation and activity is poorly defined. In this study, we investigated the hypothesis that in chronic inflammation activated T cells contribute to enhanced bone turnover by promoting osteoblastic differentiation. We show that T cells produce soluble factors that induce alkaline phosphatase activity in bone marrow stromal cells and elevated expression of mRNA for Runx2 and osteocalcin. This data indicate that T cell derived factors have the capacity to stimulate the differentiation of bone marrow stromal cells into the osteoblast phenotype. RANKL mRNA was undetectable under any conditions in highly purified bone marrow stromal cells. In contrast, RANKL was constitutively expressed in primary osteoblasts and only moderately up-regulated by activated T cell conditioned medium. Interestingly, both bone marrow stromal cells and osteoblasts expressed mRNA for RANK, which was strongly up-regulated in both cell types by activated T cell conditioned medium. Although, mRNA for the RANKL decoy receptor, osteoprotegerin, was also up-regulated by activated T cell conditioned medium, it's inhibitory effects may be mitigated by a simultaneous rise in the osteoprotegerin competitor TNF-related apoptosis-inducing ligand. Based on our data we propose that during chronic inflammation, T cells regulate bone loss by a dual mechanism involving both direct stimulation of osteoclastogenesis, by production of osteoclastogenic cytokines, and indirectly by induction of osteoblast differentiation and up-regulation of bone turnover via coupling.     

Mechanical strain differentially regulates endothelial nitric-oxide synthase and receptor activator of nuclear kappa B ligand expression via ERK1/2 MAPK

Exercise promotes positive bone remodeling through controlling cellular processes in bone. Nitric oxide (NO), generated from endothelial nitric-oxide synthase (eNOS), prevents resorption, whereas receptor activator of nuclear kappa B ligand (RANKL) promotes resorption through regulating osteoclast activity. Here we show that mechanical strain differentially regulates eNOS and RANKL expression from osteoprogenitor stromal cells in a magnitude-dependent fashion. Strain (0.25-2%) induction of eNOS expression was magnitude-dependent, reaching a plateau at 218 +/- 36% of control eNOS. This was accompanied by increases in eNOS protein and a doubling of NO production. Concurrently, 0.25% strain inhibited RANKL expression with increasing response up to 1% strain (44 +/- 3% of control RANKL). These differential responses to mechanical input were blocked when an ERK1/2 inhibitor was present during strain application. Inhibition of NO generation did not prevent strain-activated ERK1/2. To confirm the role of ERK1/2, cells were treated with an adenovirus encoding a constitutively activated MEK; Ad.caMEK significantly increased eNOS expression and NO production by more than 4-fold and decreased RANKL expression by half. In contrast, inhibition of strain-activated c-Jun kinase failed to prevent strain effects on either eNOS or RANKL. Our data suggest that physiologic levels of mechanical strain utilize ERK1/2 kinase to coordinately regulate eNOS and RANKL in a manner leading to positive bone remodeling.     

Critical role of filamin-binding LIM protein 1 (FBLP-1)/migfilin in regulation of bone remodeling

Bone remodeling is a complex process that must be precisely controlled to maintain a healthy life. We show here that filamin-binding LIM protein 1 (FBLP-1, also known as migfilin), a kindlin- and filamin-binding focal adhesion protein, is essential for proper control of bone remodeling. Genetic inactivation of FBLIM1 (the gene encoding FBLP-1) in mice resulted in a severe osteopenic phenotype. Primary FBLP-1 null bone marrow stromal cells (BMSCs) exhibited significantly reduced extracellular matrix adhesion and migration compared with wild type BMSCs. Loss of FBLP-1 significantly impaired the growth and survival of BMSCs in vitro and decreased the number of osteoblast (OB) progenitors in bone marrow and OB differentiation in vivo. Furthermore, the loss of FBLP-1 caused a dramatic increase of osteoclast (OCL) differentiation in vivo. The level of receptor activator of nuclear factor κB ligand (RANKL), a key regulator of OCL differentiation, was markedly increased in FBLP-1 null BMSCs. The capacity of FBLP-1 null bone marrow monocytes (BMMs) to differentiate into multinucleated OCLs in response to exogenously supplied RANKL, however, was not different from that of WT BMMs. Finally, we show that a loss of FBLP-1 promotes activating phosphorylation of ERK1/2. Inhibition of ERK1/2 activation substantially suppressed the increase of RANKL induced by the loss of FBLP-1. Our results identify FBLP-1 as a key regulator of bone homeostasis and suggest that FBLP-1 functions in this process through modulating both the intrinsic properties of OB/BMSCs (i.e., BMSC-extracellular matrix adhesion and migration, cell growth, survival, and differentiation) and the communication between OB/BMSCs and BMMs (i.e., RANKL expression) that controls osteoclastogenesis.     

RANKL与骨重建

骨是一种动态更新的组织,它不断进行骨吸收（bone resorption）与骨形成（bone formation）的平衡,这个过程称之为骨重建（bone remodeling）．核因子κB受体活化因子配体（receptor activator of nuclear factor κB ligand,RANKL）是骨吸收和骨形成耦联的关键,具有诱导破骨细胞（osteoclast, OC）生成、活化,抑制破骨细胞凋亡的作用．RANKL最初发现于活化的T细胞,但骨重建过程中RANKL主要来源于骨细胞、成骨细胞和骨髓基质细胞．RANKL/核因子κB受体活化因子（receptor activator of nuclear factor κB,RANK）/骨保护素（osteoprotegerin, OPG）信号通路在成骨细胞调控破骨细胞生成的过程中起着重要的调节作用,是维持骨重建平衡的关键．本文就RANKL及其在骨中的分子作用机制作一综述.     

Sphingosine 1-phosphate as a regulator of osteoclast differentiation and osteoclast-osteoblast coupling

Sphingosine 1-phosphate (S1P), produced by sphingosine kinase (SPHK), acts both by intracellular and extracellular modes. We evaluated the role of SPHK1 and S1P in osteoclastogenesis using bone marrow-derived macrophage (BMM) single and BMM/osteoblast coculture systems. In BMM single cultures, the osteoclastogenic factor receptor activator of NF-kappaB ligand (RANKL) upregulated SPHK1 and increased S1P production and secretion. SPHK1 siRNA enhanced and SPHK1 overexpression attenuated osteoclastogenesis via modulation of p38 and ERK activities, and NFATc1 and c-Fos levels. Extracellular S1P had no effect in these cultures. These data suggest that intracellular S1P produced in response to RANKL forms a negative feedback loop in BMM single cultures. In contrast, S1P addition to BMM/osteoblast cocultures greatly increased osteoclastogenesis by increasing RANKL in osteoblasts via cyclooxygenase-2 and PGE(2) regulation. S1P also stimulated osteoblast migration and survival. The RANKL elevation and chemotactic effects were also observed with T cells. These results indicate that secreted S1P attracts and acts on osteoblasts and T cells to augment osteoclastogenesis. Taken together, S1P plays an important role in osteoclastogenesis regulation and in communication between osteoclasts and osteoblasts or T cells.     

RANKL from bone marrow adipose lineage cells promotes osteoclast formation and bone loss

Receptor activator of NF‐κB ligand (RANKL) is essential for osteoclast formation and bone remodeling. Nevertheless, the cellular source of RANKL for osteoclastogenesis has not been fully uncovered. Different from peripheral adipose tissue, bone marrow (BM) adipose lineage cells originate from bone marrow mesenchymal stromal cells (BMSCs). Here, we demonstrate that adiponectin promoter‐driven Cre expression (Adipoq^Cre ) can target bone marrow adipose lineage cells. We cross the Adipoq^Cre mice with rankl^fl/fl mice to conditionally delete RANKL from BM adipose lineage cells. Conditional deletion of RANKL increases cancellous bone mass of long bones in mice by reducing the formation of trabecular osteoclasts and inhibiting bone resorption but does not affect cortical bone thickness or resorption of calcified cartilage. Adipoq^Cre; rankl^fl/fl mice exhibit resistance to estrogen deficiency and rosiglitazone (ROS)‐induced trabecular bone loss but show bone loss induced by unloading. BM adipose lineage cells therefore represent an essential source of RANKL for the formation of trabecula osteoclasts and resorption of cancellous bone during remodeling under physiological and pathological conditions. Targeting bone marrow adiposity is a promising way of preventing pathological bone loss.     

Expression of RANKL/OPG during bone remodeling in vivo

The interaction between receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) plays a dominant role in osteoclastogenesis. As both proteins are produced by osteoblast lineage cells, they are considered to represent a key link between bone formation and resorption. In this study, we investigated the expression of RANKL and OPG during bone remodeling in vivo to determine the relationship between osteoclastogenic stimulation and osteoblastic differentiation.Total RNA was prepared from rat femurs after marrow ablation on days 0, 3, 6, and 9. The temporal activation patterns of osteoblast-related genes (procollagen α1 (I), alkaline phosphatase, osteopontin, and osteocalcin) were examined by Northern blot analysis. An appreciable increase in the expression of these osteoblast markers was observed on day 3. The peak increase in gene expression was observed on day 6 followed by a slight reduction by day 9. Real-time PCR analysis showed that the OPG mRNA expression was markedly upregulated on day 6 and slightly decreased on day 9. In contrast, RANKL mRNA expression was increased by more than 20-fold on day 9. The RANKL/OPG ratio, an index of osteoclastogenic stimulation, peaked on day 9. Histological analysis showed that RANKL and OPG immunoreactivity were predominantly associated with bone marrow cells. The expression of bone formation markers was activated in the bone formation phase, followed by the stimulation of RANKL/OPG expression in the bone resorption phase, which confirmed that these molecules are key factors linking bone formation to resorption during bone remodeling.     

Modeled microgravity stimulates osteoclastogenesis and bone resorption by increasing osteoblast RANKL/OPG ratio

Mechanical unloading causes detrimental effects on the skeleton, but the underlying mechanisms are still unclear. We investigated the effect of microgravity on osteoblast ability to regulate osteoclastogenesis. Mouse osteoblast primary cultures were grown for 24 h at unit gravity or under simulated microgravity, using the NASA-developed Rotating Wall Vessel bioreactor. Conditioned media (CM) from osteoblasts subjected to microgravity increased osteoclastogenesis and bone resorption in mouse bone marrow cultures. In these osteoblasts, the RANKL/OPG ratio was higher relative to 1g. Consistently, treatment with high concentrations of OPG-inhibited osteoclastogenesis and bone resorption in the presence of CM arising from osteoblasts cultured under microgravity. Microgravity failed to affect osteoblast differentiation and function in the time frame of the experiment, as we found no effect on alkaline phosphatase mRNA and activity, nor on Runx2, osteocalcin, osteopontin, and collagen1A2 mRNA expression. In contrast, microgravity induced a time dependent increase of ERK-1/2 phosphorylation, while phospho-p38 and phospho-JNK remained unchanged. Apoptosis, revealed by bis-benzimide staining, was similar among the various gravity conditions, while it was increased under microgravity after treatment with the MEK-1/2 inhibitor, PD98059, suggesting a protection role by ERK-1/2 against cell death. In conclusion, microgravity is capable to indirectly stimulate osteoclast formation and activity by regulating osteoblast secretion of crucial regulatory factors such as RANKL and OPG. We hypothesize that this mechanism could contribute to bone loss in individuals subjected to weightlessness and other unloading conditions.     

RANK ligand expression in heat shock factor-2 deficient mouse bone marrow stromal/preosteoblast cells

Heat Shock Proteins (HSP) are molecular chaperones activated upon cellular stress/stimuli. HSP gene expression is regulated by Heat Shock Factors (HSF). We have recently demonstrated a functional role for heat shock factor-2 (HSF-2) in fibroblast growth factor-2 (FGF-2)-induced RANK ligand (RANKL), a critical osteoclastogenic factor expression on stromal/preosteoblast cells. In the present study, we show that FGF-2 treatment did not induce RANKL expression in HSF-2-/-stromal/preosteoblast cells. Interestingly, HSF-2 deficiency resulted in rapid induction of alkaline phosphatase (ALP) activity and osteocalcin mRNA expression in these cells. Furthermore, FGF-2 did not induce osteoclast formation in co-culture of normal mouse spleen cells and HSF-2-/-stromal/preosteoblast cells. Electron microscopy analysis demonstrated that osteoclasts from HSF-2-/-mice have poorly developed ruffled borders. These data further confirm that HSF-2 plays an important role in FGF-2-induced RANKL expression in stromal/preosteoblast cells. HSF-2 deficiency has pleotropic effects on gene expression during osteoblast differentiation and osteoclastogenesis in the bone microenvironment. Novel therapeutic agents that modulate HSF-2 activation may have therapeutic utility against increased levels of FGF-2 and bone destruction associated with pathologic conditions.     

STAT3 activation in stromal/osteoblastic cells is required for induction of the receptor activator of NF-kappaB ligand and stimulation of osteoclastogenesis by gp130-utilizing cytokines or interleukin-1 but not 1,25-dihydroxyvitamin D3 or parathyroid hormone.

Interleukin (IL)-6-type cytokines stimulate osteoclastogenesis by activating gp130 in stromal/osteoblastic cells and may mediate some of the osteoclastogenic effects of other cytokines and hormones. To determine whether STAT3 is a downstream effector of gp130 in the osteoclast support function of stromal/osteoblastic cells and whether the gp130/STAT3 pathway is utilized by other osteoclastogenic agents, we conditionally expressed dominant negative (dn)-STAT3 or dn-gp130 in a stromal/osteoblastic cell line (UAMS-32) that supports osteoclast formation. Expression of either dominant negative protein abolished osteoclast formation stimulated by IL-6 + soluble IL-6 receptor, oncostatin M, or IL-1 but not by parathyroid hormone or 1,25-dihydroxyvitamin D3. Because previous studies suggested that IL-6-type cytokines may stimulate osteoclastogenesis by inducing expression of the tumor necrosis factor-related protein, receptor activator of NF-kappaB ligand (RANKL), we conditionally expressed RANKL in UAMS-32 cells and found that this was sufficient to stimulate osteoclastogenesis. Moreover, dn-STAT3 blocked the ability of either IL-6 + soluble IL-6 receptor or oncostatin M to induce RANKL. These results establish that STAT3 is essential for gp130-mediated osteoclast formation and that the target of STAT3 during this process is induction of RANKL. In addition, this study demonstrates that activation of the gp130-STAT3 pathway in stromal/osteoblastic cells mediates the osteoclastogenic effects of IL-1, but not parathyroid hormone or 1, 25-dihydroxyvitamin D3.     

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.     

Evidence for osteocyte regulation of bone homeostasis through RANKL expression

Osteocytes embedded in bone have been postulated to orchestrate bone homeostasis by regulating both bone-forming osteoblasts and bone-resorbing osteoclasts. We find here that purified osteocytes express a much higher amount of receptor activator of nuclear factor-κB ligand (RANKL) and have a greater capacity to support osteoclastogenesis in vitro than osteoblasts and bone marrow stromal cells. Furthermore, the severe osteopetrotic phenotype that we observe in mice lacking RANKL specifically in osteocytes indicates that osteocytes are the major source of RANKL in bone remodeling in vivo.     

RANKL-induced schlafen2 is a positive regulator of osteoclastogenesis

Osteoclasts are hematopoietic lineage derived-multinucleated cells that resorb bone. Their activity in balance with that of osteoblast is essential for bone homeostasis. Receptor activator of NF-κB ligand (RANKL) is known as an essential cytokine for the osteoclastogenesis, and c-Jun signaling in cooperation with NFAT family is crucial for RANKL-regulated osteoclastogenesis. We show here that schlafen2 (Slfn2), a member of a new family of growth regulatory genes involved in thymocyte development, is critical for osteoclastogenesis. RANKL selectively induces Slfn2 expression in osteoclast precursors via Rac1 signaling pathway. Targeted inhibition of Slfn2 by small interfering RNAs (siRNAs) markedly inhibits the formation of osteoclasts by diminishing the activation of c-Jun and the expression of c-Jun and NFATc1. In contrast, the overexpression of Slfn2 markedly increased phosphorylation and transactivation of c-Jun by RANKL. Together, these results indicate that Slfn2 has an essential role in osteoclastogenesis, functioning upstream of c-Jun and NFATc1.     

Curcumin inhibits osteoclastogenesis by decreasing receptor activator of nuclear factor-kappaB ligand (RANKL) in bone marrow stromal cells

Curcumin (diferuloylmethane), a pigment derived from turmeric, has anti-oxidant and anti-inflammatory activities. Accumulating evidence points to a biochemical link between increased oxidative stress and reduced bone density. Osteoclast formation was evaluated in co-cultures of bone marrow stromal cells (BMSC) and whole bone marrow cells (BMC). Expression of receptor activator of nuclear factor-kappaB ligand (RANKL) was analyzed at the mRNA and protein levels. Exposure to curcumin led to dose-dependent suppression of osteoclastogenesis in the coculture system, and to reduced expression of RANKL in IL-1alpha-stimulated BMSCs. Addition of RANKL abolished the inhibition of osteoclastogenesis by curcumin, whereas the addition of prostaglandin E2(PGE2) did not. The decreased osteoclastogenesis induced by curcumin may reduce bone loss and be of potential benefit in preventing and/or attenuating osteoporosis.