Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families.

Osteoblasts/stromal cells are essentially involved in osteoclast differentiation and function through cell-to-cell contact (Fig. 8). Although many attempts have been made to elucidate the mechanism of the so-called "microenvironment provided by osteoblasts/stromal cells," (5-8) it has remained an open question until OPG and its binding molecule were cloned. The serial discovery of the new members of the TNF receptor-ligand family members has confirmed the idea that osteoclast differentiation and function are regulated by osteoblasts/stromal cells. RANKL, which has also been called ODF, TRANCE, or OPGL, is a member of the TNF ligand family. Expression of RANKL mRNA in osteoblasts/stromal cells is up-regulated by osteotropic factors such as 1 alpha, 25(OH)2D3, PTH, and IL-11. Osteoclast precursors express RANK, a TNF receptor family member, recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into pOCs in the presence of M-CSF. RANKL is also involved in the survival and fusion of pOCs and activation of mature osteoclasts. OPG, which has also been called OCIF or TR1, is a soluble receptor for RANKL and acts as a decoy receptor in the RANK-RANKL signaling system (Fig. 8). In conclusion, osteoblasts/stromal cells are involved in all of the processes of osteoclast development, such as differentiation, survival, fusion, and activation of osteoclasts (Fig. 8). Osteoblasts/stromal cells can now be replaced with RANKL and M-CSF in dealing with the whole life of osteoclasts. RANKL, RANK, and OPG are three key molecules that regulate osteoclast recruitment and function. Further studies on these key molecules will elucidate the molecular mechanism of the regulation of osteoclastic bone resorption. This line of studies will establish new ways to treat several metabolic bone diseases caused by abnormal osteoclast recruitment and functions such as osteopetrosis, osteoporosis, metastatic bone disease, Paget's disease, rheumatoid arthritis, and periodontal bone disease.     

Inhibition of rat cardiac fibroblast growth by cAMP – but not by cGMP-dependent protein kinase

Cardiac fibroblasts play a critical role in the process of pathophysiological cardiac hypertrophy as the cell type responsible for fibrosis. Whereas many growth factors and hormones are thought to be involved, possible crosstalks between signal transduction pathways are not well defined. Therefore we investigated the influence of cAMP- and cGMP-dependent protein kinases (cAK, CGK) on platelet derived growth factor (PDGF) stimulated growth of primary cardiac fibroblasts from adult rats. We show here that PDGF-BB induced cell proliferation can be inhibited by activation of the endogenous cAK directly via the cAMP analog 5,6-DCL-cBIMPS as well as indirectly via the cAMP-elevating receptor agonist prostaglanding-E₁ (PGE1). In contrast, activation of the endogenous cGK-I has no influence on cardiac fibroblast cell growth. The strength of the proliferation inhibition is dependent on the time course of cAK activation, i. e., longer activation with the cAMP analog results in stronger proliferation inhibition. No significant influence of cAK or cGK-I on Akt activation or on the short-term activation of the MAPK cascade was observed. In contrast, 5,6-DCI-cBIMPS treatment of cardiac fibroblasts causes an inhibition of long-term MAPK phosphorylation. A prolonged PGE1-dependent cAMP signal after addition of 3-isobutyl-1-methyl-xanthine (IBMX) blocks the second long-term MAPK phosphorylation as well. Therefore the suppression of MAPK long-term phosphorylation, four to eight hours after PDGF-BB stimulation, appears to play the major role in inhibition of cardiac fibroblast proliferation by cAK activators. Received: 26 June 2000, Returned for revision: 21 July 2000, Revision received: 27 September 2000, Accepted: 11 October 2000     

Bone loss in inflammatory arthritis: mechanisms and treatment strategies

PURPOSE OF REVIEW: Focal bone loss in inflammatory arthritis begins early in the disease process and can contribute to patient morbidity. Current treatment strategies primarily target suppression of the inflammatory cascade with varying success in limiting the progression of focal bone destruction. This review outlines the current understanding of the mechanisms mediating inflammation-induced focal bone loss in rheumatoid arthritis and other inflammatory arthritides and highlights recent studies in animal models of arthritis that have contributed to our knowledge of this process. RECENT FINDINGS: Bone-resorbing osteoclasts have been identified as important effector cells in inflammation-induced bone loss in both experimental animal models and human rheumatoid arthritis and psoriatic arthritis. The RANK/RANKL (receptor activator of nuclear factor-kappaB and RANK ligand) pathway has been shown to be essential for osteoclast differentiation in inflammatory arthritis. In addition, in vitro and in vivo studies have demonstrated that many cytokines and growth factors elaborated by inflamed synovial tissues may contribute to osteoclast differentiation and activation. SUMMARY: Elucidation of the mechanisms mediating osteoclast differentiation and function has identified new pathways for potential targeted therapeutic intervention for focal bone loss in inflammatory arthritis. Challenges in the application of this approach are that therapies targeting the osteoclast would need to be used in combination with effective anti-inflammatory agents, and that pathways mediating osteoclast differentiation and function would need to remain at least partially functional to allow for continued skeletal remodeling.     

Mechanisms involved in bone resorption

Osteoclasts, which are present only in bone, are multinucleated giant cells with the capacity to resorb mineralized tissues. These osteoclasts are derived from hemopoietic progenitors of the monocyte-macrophage lineage. Osteoblasts or bone marrow-derived stromal cells are involved in osteoclastogenesis through a mechanism involving cell-to-cell contact with osteoclast progenitors. Experiments on the osteopetrotic op/op mouse model have established that a product ofosteo blasts, macrophage colony-stimulating factor (M-CSF), regulates differentiation of osteoclast progenitors into osteoclasts. Recent discovery of osteoclast differentiation factor (ODF)/receptor activator of NF-κ Bligand (RANKL) allowed elucidation of the precise mechanism by which osteoblasts regulate osteoclastic bone resorption. Treatment of osteoblasts with bone-resorbing factors up-regulated expression of RANKL mRNA. In contrast, TNF α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the RANKL system. IL-1 also directly acts on mature osteoclasts as a potentiator of osteoclast activation. In addition, TGF-β super family members, such as bone morphogenetic proteins(BMPs) strikingly enhanced osteoclast differentiation from their progenitors and survival of mature osteoclasts induced by RANKL. These results suggest that BMP-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and function. This revised version was published online in July 2006 with corrections to the Cover Date.     

New insight in the mechanism of osteoclast activation and formation in multiple myeloma: focus on the receptor activator of NF-kappaB ligand (RANKL)

The increase of osteoclast activation and formation is mainly involved in the development of the osteolytic bone lesions that characterize multiple myeloma (MM) patients. The mechanisms by which myeloma cells induce bone resorption have not been clear for many years. Recently, new evidence has elucidated which factors are critically involved in the activation of osteoclastic cells in MM. The potential role of the critical osteoclastogenic factor, the receptor activator of NF-kappaB ligand (RANKL), and its soluble antagonist osteoprotegerin (OPG) in the activation of bone resorption in MM is summarized in this review. It has been demonstrated that human MM cells induce an imbalance in the bone marrow environment of the RANKL/OPG ratio in favor of RANKL that triggers the osteoclast formation and activation leading to bone destruction. The direct production of the chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha) by myeloma cells, in combination with the RANKL induction in BM stromal cells in response to myeloma cells, are critical in osteoclast activation and osteoclastogenesis.     

Treatment strategies for bone disease

Multiple myeloma is characterized by extensive bone destruction with little or no new bone formation. A multiplicity of factors including receptor activator NF-kappaB (RANKL), macrophage inflammatory protein-1alpha, interleukin-3 and interleukin-6 can induce osteoclast formation in myeloma and drive the bone destructive process. Furthermore, factors are also produced either in the microenvironment or by myeloma cells themselves, which inhibit osteoblast differentiation and new bone formation. The combination of increased osteoclast formation with little or no bone repair in response to the previous bone destruction explains the severity of the bone disease in myeloma. Studies of the pathophysiology of myeloma bone disease have identified several novel therapeutic targets. These include antibodies to RANKL, chemokine receptor antagonists, which block the effects of chemokines on osteoclast differentiation and proteasome antagonists, which can affect both RANKL production and osteoprotegerin levels as well as inhibit osteoclast and enhance osteoblast differentiation. In addition, many of the new biologic agents being used for the treatment of patients with myeloma also further inhibit the bone destructive process. New therapies that can target both the tumor as well as the severe bone disease should be on the horizon to treat this devastating complication of myeloma.     

Rank/Rankl/opg: literature review

The discovery of the receptor activator of nuclear factor-kB (RANK)/RANK Ligand (RANKL)/osteoprotegerin (OPG) pathway contributed to the understanding of how bone formation and resorption were processed and regulated. RANKL and OPG are members of the tumor necrosis factor (TNF) and TNF receptor (TNFr) superfamilies, respectively, and binding to receptor activator of NF-kB (RANK) not only regulate osteoclast formation, activation and survival in normal bone modeling and remode-ling, but also in several other pathologic conditions characterized by increased bone turnover. There is accumulating evidence of the potential role of OPG and RANKL in other tissues. Looking beyond the RANK/RANKL/OPG axis, Wingless (Wnt) pathway emerged as the osteoblast differentiation way, and also as a bone mass regulator. Researchers have been discovering new molecules and cytokines interactions. Altogether, data suggest that RANK/RANKL/OPG system could be targeted as a new treatment strategy in bone conditions. FREEDOM is the more recently published clinical trial about a RANKL-specific recombinant fully human monoclonal antibody (denosumab). OPG is also a potential innovative therapeutic option to be investigated.     

Osteoprotegerin (OPG/OCIF) inhibits bone destruction in rheumatoid arthritis models

Recent discovery of factors involved in bone destruction in Rheumatoid Arthritis (RA) identified its molecular mechanism. Osteoclast differentiation factor (ODF, also called receptor activator of NF-kappaB ligand (RANKL) ) that controls osteoclast differentiation and function has a major role in the bone destruction among them. Osteoclastogenesis inhibitory factor (OCIF, also called osteoprotegerin (OPG) ) that is a decoy receptor for ODF/RANKL is a specific inhibitor of bone destruction. OPG/OCIF may be useful for and applicable to the treatment of bone destruction in RA.     

Handgrip Strength is an Independent Predictor of Distal Radius Bone Mineral Density in Postmenopausal Women

Several cross-sectional studies have reported a positive correlation between muscle strength and local bone mineral density. However, very few studies have evaluated the possible role of confounding variables, which may be substantial as both bone mineral density and muscle strength are multifactorial variables. We studied 140 postmenopausal women who underwent their first osteodensitometry in our hospital. Of these, 102 women affected neither by bone diseases apart from primary osteoporosis nor treated with drugs affecting bone mass were selected. Distal radius bone mineral density of the non-dominant arm was assessed by dual photon absorptiometry. Handgrip strength was measured by a handheld dynamometer. The following factors influencing bone mass were also considered: age, years since menopause, years of cyclic ovarian activity, body weight, body height, body mass index, and both calcium and alcohol dietary intake. Statistical evaluation was performed by stepwise multiple regression analysis. This showed that only two variables were independently related to bone mineral density: handgrip strength (which was the best bone density predictor among the studied independent variables) and years since menopause. R² value was 0.43 (F=38.04, p<0.001). All the other variables studied were not significantly related to bone density when the effects of both strength and years since menopause were considered. In conclusion, the data showed that handgrip strength was a strong independent predictor of distal radius bone mineral density in postmenopausal women. Clinical assessment of osteoporosis risk factors, including muscle strength, is recommended: although it is not an adequate substitute for bone densitometry, it can help clinicians to identify the risk groups at which to direct bone density measurement. Received: 1 October 1999 / Accepted: 29 May 2000     

Apoptosis in the vascular wall and atherosclerosis

Apoptosis, programmed cell death, has emerged as a key element in the complex pathophysiology underlying the development as well as the progression of atherosclerosis. A number of recent reports provided evidence for both in vivo and in vitro occurrence of apoptotic cell death of vascular cells, namely endothelial cells, macrophages, and vascular smooth muscle cells. In addition, functional studies in disease models underscore the relevance of these findings for the understanding of processes which lead to lesion development, plaque rupture, and thrombus formation. Pathomechanistic in vitro investigations provided an increasingly detailed picture of the involved intracellular signaling pathways that regulate onset and execution of apoptosis. These insights offer the potential of therapeutic interventions targeted to interfere with the molecular processes involving apoptotic cell death in the vascular wall. Received: 18 August 2000, Returned for revision: 30 August 2000, Revision received: 11 September 2000, Accepted: 13 September 2000     

New insights into the pathophysiology and management of osteoporosis in patients with beta thalassaemia

Osteoporosis represents an important cause of morbidity in adult patients with thalassaemia major (TM). The pathogenesis of osteoporosis in TM is multifactorial, and includes bone marrow expansion, endocrine dysfunction and iron overload. Additional genetic factors, such as the COLIA 1 gene polymorphism, seem to play an important role in the development of low bone mass in these patients. However, the mechanisms through which these factors lead to bone loss have not been completely clarified. The diminished osteoblast function is accompanied by a comparable or even greater increase in osteoclast activity. The receptor activator of nuclear factor-kappa B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) pathway has been recently recognized as the final, dominant mediator of osteoclast proliferation and activation. There is increased evidence that this pathway interferes in the pathogenesis of thalassaemia-induced osteoporosis. Currently, bisphosphonates that are potent inhibitors of osteoclast function have been used in TM patients with encouraging results. This review attempts to summarize all the novel data for the biology of bone damage in TM. It also describes the results of all major studies that have investigated the effects of different treatment modalities for TM-induced osteoporosis, their mode of action, and the future implications of their use.     

Clinical utility of selected disease activity markers in patients with systemic lupus erythematosus

The objective of this study was to assess the utility of measurement of thrombomodulin, antinucleosome antibodies, sVCAM-1, sICAM-1, neopterin, fas ligand, IL-10 and sIL-2R in patients with systemic lupus erythematosus (SLE) and to compare them with traditional markers of SLE activity (anti-dsDNA antibodies, C3, C4) and the ECLAM index of disease activity. The measurement was performed over a 6-month period at three consecutive time points after 3 months in each of the 52 patients with SLE. Anti-dsDNA antibodies, thrombomodulin, antinucleosome antibodies, sVCAM-1m sICAM-1, neopterin, fas ligand, IL-10 and sIL-2R were tested by ELISA technique, while C3, C4 components of complement were tested by nephelometry. Fas ligand and IL-10 did not correlate with the ECLAM index. The rest of the markers showed significant correlation with the disease activity index. Thrombomodulin and anti-dsDNA antibodies reflect in the best way the changing trend in disease activity. Antinucleosome antibodies seem to be a promising marker useful in early diagnosis. Soluble VCAM-1, sICAM-1, neopterin and sIL-2R are interesting molecules with a role in disease pathogenesis, but their practical utility is limited. Received: 27 September 2000 / Accepted: 31 March 2001     

Osteopetrosis: from Animal Models to Human Conditions

The term “osteopetrosis” is applied to a group of disorders characterized by an increased bone density, due to an inadequate bone resorption. A considerable part of our current knowledge on osteoclast biology is based on the study of osteopetrotic animal models. The search for mutations in these animals has unveiled many molecular mechanisms underlying osteoclast differentiation and functioning. It also supplied new candidate genes for the identification of genes involved in the human variants of this disease. All osteopetrotic genes identified so far in humans have their animal counterpart. The reverse is not true. This can partially be explained by the fact that still more than 30% of all patients suffer from osteopetrosis with an unkown molecular defect. Therefore, the studies of the osteopetrotic animal models and the correlating human osteopetrotic forms were and are still very important for our knowledge of the aetiology, prognosis and treatment of this disease. This review focuses on osteopetrotic animal models as well as human osteopetrotic conditions and their impact on osteoclast biology, pathogenesis and treatment.     

Bone morphogenetic protein 2 stimulates osteoclast differentiation and survival supported by receptor activator of nuclear factor-kappaB ligand.

Bone is a major storage site for TGFbeta superfamily members, including TGFbeta and bone morphogenetic proteins. It is believed that these cytokines are released from bone during bone resorption. Recent studies have shown that both RANKL and macrophage colony-stimulating factor are two essential factors produced by osteoblasts for inducing osteoclast differentiation. In the present study we examined the effects of bone morphogenetic protein-2 on osteoclast differentiation and survival supported by RANKL and/or macrophage colony-stimulating factor. Mouse bone marrow-derived macrophages differentiated into osteoclasts in the presence of RANKL and macrophage colony-stimulating factor. TGFbeta superfamily members such as bone morphogenetic protein-2, TGFbeta, and activin A markedly enhanced osteoclast differentiation induced by RANKL and macrophage colony-stimulating factor, although each cytokine alone failed to induce osteoclast differentiation in the absence of RANKL. Addition of a soluble form of bone morphogenetic protein receptor type IA to the culture markedly inhibited not only osteoclast formation induced by RANKL and bone morphogenetic protein-2, but also the basal osteoclast formation supported by RANKL alone. Either RANKL or macrophage colony-stimulating factor stimulated the survival of purified osteoclasts. Bone morphogenetic protein-2 enhanced the survival of purified osteoclasts supported by RANKL, but not by macrophage colony-stimulating factor. Both bone marrow macrophages and mature osteoclasts expressed bone morphogenetic protein-2 and bone morphogenetic protein receptor type IA mRNAs. An EMSA revealed that RANKL activated nuclear factor-kappaB in purified osteoclasts. Bone morphogenetic protein-2 alone did not activate nuclear factor-kappaB, but rather inhibited the activation of nuclear factor-kappaB induced by RANKL in purified osteoclasts. These findings suggest that bone morphogenetic protein-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and survival. The enhancement of RANKL-induced survival of osteoclasts by bone morphogenetic protein-2 appears unrelated to nuclear factor-kappaB activation.     

Inhibitor of DASH proteases affects expression of adhesion molecules in osteoclasts and reduces myeloma growth and bone disease

Dipeptidyl peptidase (DPP) IV activity and/or structure homologues (DASH) are serine proteases implicated in tumourigenesis. We previously found that a DASH protease, fibroblast activation protein (FAP), was involved in osteoclast-induced myeloma growth. Here we further demonstrated expression of various adhesion molecules in osteoclasts cultured alone or cocultured with myeloma cells, and tested the effects of DASH inhibitor, PT-100, on myeloma cell growth, bone disease, osteoclast differentiation and activity, and expression of adhesion molecules in osteoclasts. PT-100 had no direct effects on viability of myeloma cells or mature osteoclasts, but significantly reduced survival of myeloma cells cocultured with osteoclasts. Real-time PCR array for 85 adhesion molecules revealed upregulation of 17 genes in osteoclasts after coculture with myeloma cells. Treatment of myeloma/osteoclast cocultures with PT-100 significantly downregulated 18 of 85 tested genes in osteoclasts, some of which are known to play roles in tumourigenesis and osteoclastogenesis. PT-100 also inhibited osteoclast differentiation and subsequent pit formation. Resorption activity of mature osteoclasts and differentiation of osteoblasts were not affected by PT-100. In primary myelomatous severe combined immunodeficient (SCID)-hu mice PT-100 reduced osteoclast activity, bone resorption and tumour burden. These data demonstrated that DASH proteases are involved in myeloma bone disease and tumour growth.     

Load-induced changes in repolarization: evidence from experimental and clinical data

The high incidence of arrhythmias in patients with heart failure, hypertension, valvular heart disease, or mitral walve prolapse suggests a strong link between wall motion abnormalities and arrhythmias. A potential common mechanism underlying these observations may be that overload leads to electrophysiologic changes and facilitates arrhythmias. This article summarizes the interaction between changes in atrial and ventricular loading conditions and repolarization. Most experimental and clinical studies demonstrated 1) a reduction of action potential duration and refractoriness, 2) development of early afterdepolarizations, and 3) ectopic beats originating from these afterdepolarizations. Discrepancies between studies are related to different study designs, i.e., varying magnitude, velocity, and timing of increased load, the level of repolarization at which action potential duration is measured as well as different animal species. Direct effects of increased load on repolarization are most likely caused by activation of stretch-activated nonselective cation ion channels and changes in calcium handling. Current antiarrhythmic drug therapy is aimed at electrical disorders as the primary cause of arrhythmias. If mechanical disorders play a central role in the genesis of cardiac arrhythmias, future treatment should be directed at restoring a more normal mechanical function of the heart. Additional studies will further clarify the nature and clinical significance of load-related changes in repolarization and arrhythmogenesis. Received: 20 November 2000 / Returned for revision: 20 December 2000 / Revision received: 2 January 2001 / Accepted: 8 January 2001     

Alcohol and the risk of myocardial infarction

Epidemiological studies have repeatedly demonstrated a beneficial effect of moderate alcohol consumption on the incidence of coronary heart disease, myocardial infarction and overall mortality. The latter increases with excessive alcohol consumption. Although most epidemiological studies demonstrate a beneficial effect of alcohol consumption independent from the specific kind of alcoholic beverage, there is increasing evidence that wine and in particular red wine might contain pharmacological substances, which prevent atherosclerosis and myocardial infarction independent from the wine ethanol. Pathophysiological mechanisms mediating these beneficial effects include effects of wine phenols and tannins on LDL-cholesterol oxidation status, thrombocyte aggregation, endothelial function and smooth muscle cell proliferation. Identification and characterization of the pharmacologically active substances might provide the stage for the development of new substances to be used in the prevention of coronary artery disease and myocardial infarction. Received: 14 August 2000, Returned for 1. revision: 6 September 2000, 1. Revision received: 23 November 2000, Returned for 2. revision: 5 December 2000, 2. Revision received: 21 December 2000, Accepted: 8 January 2001     

Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand

A receptor that mediates osteoprotegerin ligand (OPGL)-induced osteoclast differentiation and activation has been identified via genomic analysis of a primary osteoclast precursor cell cDNA library and is identical to the tumor necrosis factor receptor (TNFR) family member RANK. The RANK mRNA was highly expressed by isolated bone marrow-derived osteoclast progenitors and by mature osteoclasts in vivo. Recombinant OPGL binds specifically to RANK expressed by transfected cell lines and purified osteoclast progenitors. Transgenic mice expressing a soluble RANK-Fc fusion protein have severe osteopetrosis because of a reduction in osteoclasts, similar to OPG transgenic mice. Recombinant RANK-Fc binds with high affinity to OPGL in vitro and blocks osteoclast differentiation and activation in vitro and in vivo. Furthermore, polyclonal Ab against the RANK extracellular domain promotes osteoclastogenesis in bone marrow cultures suggesting that RANK activation mediates the effects of OPGL on the osteoclast pathway. These data indicate that OPGL-induced osteoclastogenesis is directly mediated through RANK on osteoclast precursor cells.     

Bilateral tibia and fibula fractures in a patient with rheumatoid arthritis

A 52-year-old woman with rheumatoid arthritis treated with low-dose steroids developed bilateral distal tibia and fibula fractures over a 15-month period. Her bone density was within osteopenic levels. Such fractures are an unusual but increasingly recognised complication of rheumatoid disease and its treatment, although there is often diagnostic delay. Bilateral fractures are particularly rare. A high level of clinical suspicion is required for early diagnosis. Received: 19 June 2000 / Accepted: 26 January 2001     

Cell adhesion molecule in osteobrast biology

Investigation of integrins among cell adhesion molecules has revealed that bone matrix proteins including type I collagen play a pivotal role in the regulation of osteoblastic differentiation via their binding to integrins on osteoblasts. It is now suggested that cell-matrix interactions are critically involved in bone formation by osteoblasts. Cell-cell interactions among osteoblasts and hematopoietic cells are also mediated by cell adhesion molecules including integrins. Recently, it is reported that adhesion of osteoclast precursors to osteoblasts via cell adhesion molecules supports the formation of bone resorbing osteoclasts. These observations indicate that cell adhesion molecules on osteoblasts are functionally involved in bone metabolism.