Osteoprotegerin and RANKL differentially regulate angiogenesis and endothelial cell function

Osteoprotegerin (OPG) a soluble tumor necrosis factor receptor family molecule protects endothelial cells from apoptosis in vitro and promotes neovascularization in vivo. In this study, we assessed the role of OPG and its ligands, receptor activator of nuclear factor-κB ligand (RANKL) and tumor necrosis factor-related apoptosis inducing ligand (TRAIL), in microvessel formation using the rat aortic ring model of angiogenesis. OPG was found to promote a twofold increase in angiogenic sprouting in the aortic ring model, and this effect was inhibited by pre-incubation with a fivefold molar excess of either RANKL or TRAIL. While TRAIL had no effect upon angiogenesis on its own, RANKL was found to potently inhibit basal and vascular endothelial growth factor-induced angiogenesis. OPG increased the rate of endothelial cell proliferation in sprouting microvessels; in contrast, RANKL inhibited proliferation. RANKL was found to induce endothelial apoptosis at days 6, 7, and 10 in the aortic ring model and after incubation with human umbilical vein endothelial cells (HUVECs). Signaling studies showed that OPG induced ERK1/2 and Akt phosphorylation in HUVECs while RANKL had no effect. Our results indicate that OPG is a positive regulator of microvessel formation, while RANKL is an angiogenic inhibitor due to effects on regulation of endothelial cell proliferation, apoptosis, and signaling.     

Osteoprotegerin prevents glucocorticoid-induced osteocyte apoptosis in mice

The adverse skeletal effects of glucocorticoid excess are due to increased osteoclast survival, decreased production of osteoblasts, and increased apoptosis of osteoblasts and osteocytes, but it remains unknown which of these is the principle cause of the decrease in bone strength. Previous studies suggested that osteocytes contribute to bone strength independently of changes in bone mass. Administration of the receptor activator for nuclear factor κB ligand (RANKL) antagonist osteoprotegerin (OPG) rapidly decreases osteoclasts followed by a decrease in osteoblasts but should not affect the long-lived osteocytes. Therefore, to distinguish between glucocorticoid effects on osteoclasts, osteoblasts, or osteocytes, we administered glucocorticoids, alone or in combination with OPG with the fragment crystallizable region of Ig heavy chains (OPG-Fc), to mice. The suppressive effect of glucocorticoids on spinal bone mineral density, cortical thickness, and strength was prevented by OPG-Fc. OPG-Fc, with or without glucocorticoids, profoundly reduced osteoclasts, osteoblasts, and bone formation. Unexpectedly, OPG-Fc prevented the glucocorticoid-induced increase in osteocyte apoptosis and reduction in solute transport from the systemic circulation to the osteocyte-lacunar-canalicular network. The fluid in the osteocyte-lacunar-canalicular network was inversely related to osteocyte apoptosis and directly related to bone mineral density. Consistent with the in vivo findings, Both OPG-Fc and OPG decreased glucocorticoid-induced apoptosis of MLO-Y4 osteocytic cells. OPG can also bind and antagonizes the activity of the TNF-related apoptosis-inducing ligand (TRAIL), but glucocorticoids did not change TRAIL expression, and knockdown of TRAIL did not prevent OPG-Fc from reducing glucocorticoid-induced osteocyte apoptosis. Based on these results, we conclude that at least part of the OPG-induced preservation of bone strength is due to the maintenance of osteocyte viability and the lacunar-canalicular network.     

Tumor necrosis factor-related apoptosis-inducing ligand and vascular calcification

Vascular calcification is frequent in patients with chronic kidney disease. Osteoprotegerin (OPG, a soluble factor which blocks osteoclast differentiation) has recently been implicated in the genesis of vascular calcification. Given that OPG can bind the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we hypothesized that the TRAIL protein is involved in the formation of vascular calcification both in vitro and in vivo. Using an immunohistochemical approach, we evaluated TRAIL and OPG expression on aortic valves slides from non-uremic and uremic wild type and apolipoprotein knockout (Apo E(-/-) ) mice. We also tested the in vitro effects of TRAIL on cultured primary human vascular smooth muscle cells (hVSMC). We further assayed serum soluble TRAIL (sTRAIL) levels in hemodialysis patients and correlated them with vascular calcification scores. Our results demonstrated that: (i) TRAIL and OPG were expressed inside the atheroma plaque in non-uremic ApoE(-/-) mice, but not in wild type mice; and (ii) uremia enhanced the expression levels. TRAIL enhanced the phosphate-induced mineralization of hVSMCs in a dose-dependent manner. In clinical terms, we demonstrated that sTRAIL is depressed in the sera of hemodialysis patients, but was not correlated with vascular calcification. Our results suggest that TRAIL may be involved in the formation of vascular calcification in certain experimental settings; however, its role in chronic kidney disease patients requires further evaluation.     

The effect of the alendronate on OPG/RANKL system in differentiated primary human osteoblasts

Alendronate is a well-established treatment for osteoporosis and suppresses bone resorption by a direct effect on osteoclasts and their precursors. The effect of alendronate on osteoclasts is produced, at least in part, by the receptor activator of nuclear factor kappaB ligand (RANKL) and the osteoprotegerin (OPG) synthesized by the osteoblasts. This study analyzes the effect of alendronate in cell viability, alkaline phosphatase (ALP) activity and RANKL and OPG expression in primary human osteoblasts (hOB). Alendronate at concentrations lower than 10?? M did not have a toxic effect on hOB in vitro and did not modify the ALP activity at least for 72 h. Alendronate did not change OPG expression in basal, 10% fetal bovine serum (FBS), and vitamin D-treated cultures. Similar results were observed at the protein level. Unexpectedly, alendronate at 10?? and 10?? M concentrations increased the RANKL expression with the presence of vitamin D in differentiated hOB, and this induction of RANKL mRNA levels by alendronate was dose-dependent. However, this effect was not observed in basal and 10% FBS culture conditions. Thus, we conclude that alendronate does not affect the ALP activity and OPG gene expression in differentiated hOB, but may increase RANKL gene expression induced by vitamin D.     

The effect of the alendronate on OPG/RANKL system in differentiated primary human osteoblasts

Alendronate is a well-established treatment for osteoporosis and suppresses bone resorption by a direct effect on osteoclasts and their precursors. The effect of alendronate on osteoclasts is produced, at least in part, by the receptor activator of nuclear factor kappaB ligand (RANKL) and the osteoprotegerin (OPG) synthesized by the osteoblasts. This study analyzes the effect of alendronate in cell viability, phosphatase alkaline (ALP) activity and RANKL, and OPG expression in primary human osteoblasts (hOB). Alendronate at concentrations lower than 10(-5) M did not have a toxic effect on hOB in vitro and did not modify the ALP activity at least for 72 h. Alendronate did not change OPG expression in basal, 10% FBS, and vitamin D-treated cultures. Similar results were observed at the protein level. Unexpectedly, alendronate at 10(-7) and 10(-5) M concentrations increased the RANKL expression with the presence of vitamin D in differentiated hOB and this induction of RANKL mRNA levels by alendronate was dose-dependent. However, this effect was not observed in basal and 10% FBS culture conditions. Thus, we conclude that alendronate does not affect the ALP activity and OPG gene expression in differentiated hOB, but may increase RANKL gene expression induced by vitamin D.     

Cytokine-controlled RANKL and osteoprotegerin expression by human and mouse synovial fibroblasts: fibroblast-mediated pathologic bone resorption

OBJECTIVE: To determine whether proinflammatory cytokine treatment or the complete absence of select cytokines modulates the expression of RANKL and osteoprotegerin (OPG) in synovial fibroblasts. METHODS: Fibroblasts were isolated from normal and rheumatoid human synovium and from normal or arthritic joints of wild-type and cytokine gene-deficient (interleukin-4-knockout [IL-4 (-/-)] and interferon-gamma-knockout [IFNgamma (-/-)]) mice. Fibroblasts were stimulated with proinflammatory cytokines (tumor necrosis factor alpha [TNFalpha], IL-1beta, and IL-17) or antiosteoclastogenic cytokines (IL-4 and IFNgamma), alone or in combination, and the expression of RANKL and OPG was measured. RESULTS: Proinflammatory cytokine-stimulated fibroblasts from rheumatoid and arthritic mouse joints expressed higher levels of RANKL and OPG than those from normal joints. IL-4 suppressed RANKL expression and increased OPG expression, IFNgamma reduced the production of both RANKL and OPG, and IL-17 had only a modest effect on the expression of RANKL or OPG. Additive effects of combination treatment (TNFalpha/IL-17 or IL-1beta/IL-17) were observed only in the human system. Extensive destruction was observed in the arthritic joints of IL-4 (-/-) mice, with a corresponding upward shift of the RANKL:OPG ratios. However, an IL-17 deficiency did not attenuate arthritis or reduce bone resorption. CONCLUSION: Proinflammatory cytokines induce the expression of RANKL and OPG in both human and murine synovial fibroblasts. The RANKL:OPG ratios are shifted in favor of bone protection by IL-4 treatment, and, to a lesser extent, by IFNgamma treatment. Unexpectedly, an IL-17 deficiency alone does not induce reduced inflammatory bone destruction. Our results suggest that synovial fibroblasts may significantly contribute to bone resorption through modulation of RANKL and OPG production in a cytokine-rich milieu of inflamed joints.     

The relationship between four single nucleotide polymorphisms in the promoter region of the osteoprotegerin gene and aortic calcification or coronary artery disease in Koreans

OBJECTIVE: Osteoprotegerin (OPG) is a recently identified cytokine that acts as a decoy receptor for the receptor activator of the NF-kappaB ligand (RANKL). OPG has been shown to be an important inhibitor of osteoclastogenesis and arterial calcification in animal models. OPG has been proposed as a link molecule between osteoporosis and arterial calcification, but the relationship between the OPG gene and the cardiovascular system in human populations is unclear. Thus, the aim of this study was to investigate the relationship between OPG gene polymorphisms and aortic calcification or coronary artery disease in Koreans. DESIGN AND PATIENTS: Genotyping of four polymorphisms, A163G, G209A, T245G and T950C, in the promoter region of the OPG gene was performed in 251 healthy Korean women (mean age 51.3 +/- 6.9 years) and in a second study population consisting of 100 patients who underwent coronary angiography (mean age 57.0 +/- 11.9 years), by allelic discrimination using the 5' nuclease polymerase chain reaction assay. Cardiovascular risk factors and serum OPG levels were measured and aortic calcification in thoracic and abdominal aorta was examined by simple radiological methods. RESULTS: In the first study population, the prevalence of aortic calcification increased significantly as the subjects grew older. The frequencies of mutant alleles were significantly higher in the subjects with aortic calcification compared with those without aortic calcification in G209A and T950C polymorphisms, although these significances were lost after adjustment for age. No significant relationship was found between OPG gene polymorphisms and serum OPG levels or cardiovascular risk factors. In the second study group, there were no associations between OPG promoter genotypes and aortic calcification, serum OPG levels, or coronary artery disease. CONCLUSIONS: We observed that the four polymorphisms in the promoter region of the OPG gene were not associated with aortic calcification or coronary artery disease in Koreans. Further studies are needed to clarify this relationship.     

Interleukin-6 (IL-6), IL-1, receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin production by human osteoblastic cells: comparison of the effects of 17-beta oestradiol and raloxifene

Oestrogen inhibits bone resorption, at least in part, by regulating the production of several cytokines, including interleukin-6 (IL-6), IL-1, receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) by cells of the osteoblastic lineage. The selective oestrogen receptor modulator raloxifene (RAL) acts on bone in a similar manner to oestrogen, although the mechanisms of action of RAL on osteoblasts still remain unclear. We investigated and compared the effects of 17-beta oestradiol (E(2)) and RAL on the regulation of IL-6, IL-1, RANKL and OPG in vitro in primary human osteoblastic (HOB) cells and in an immortalised clonal human bone marrow stromal cell line (HCC1) with osteoblastic characteristics. We tested E(2) and RAL at concentrations ranging from 10(-12) to 10(-6) M. IL-6, IL-1alpha and IL-1beta, OPG and RANKL were measured by ELISA. RANKL and OPG mRNA steady state level was assessed by quantitative PCR analysis. Both E(2) and RAL led to a significant reduction in IL-6 production in the HOB cells, although the effect was more marked with E(2) (P<0.05). IL-1alpha and IL-1beta also decreased significantly following treatment with E(2) and RAL in the HCC1 cells (E(2) (10(-8), 10(-7) and 10(-6) M), % reduction (means+/-S.E.M.) compared with vehicle-treated cells - IL-1alpha: 84+/-7.4, 70.8+/-2.9*, 78.2+/-4.8*; IL-1beta: 79+/-10, 72.8+/-8.2*, 66.6+/-2.8*; RAL (10(-8), 10(-7) and 10(-6) M) - IL-1alpha: 72.4+/-5*, 79+/- 5.2*, 102+/-7.7; IL-1beta: 67.9+/-3.2*, 69+/-2.5*, 73.8+/- 6.2*; *P<0.05). OPG protein concentration decreased significantly in a dose-dependent manner following treatment with E(2) and RAL (% reduction E(2) (10(-8), 10(-7) and 10(-6) M) - HOB: 72.5+/-8.4*, 80+/-6.7*, 62.8+/-8.9*; HCC1: 109+/-4, 98.8+/-6, 54.5+/-3.4*; RAL (10(-8), 10(-7) and 10(-6) M) - HOB: 81.5+/-5.5*, 62.7+/-7.4*, 55.2+/-10.9*; HCC1: 92.7+/-7.4, 67+/-12.2*, 39+/-4.5*; *P<0.05). In the HCC1 cells, RANKL protein did not change significantly following E(2). In contrast, a significant reduction in RANKL was seen with RAL at 10(-7) and 10(-6) M (66+/-6.4% and 74+/-3% respectively). There was no change in OPG mRNA expression following E(2) or RAL in the HCC1 cells, although in the HOB cells we observed a significant reduction in OPG mRNA. RANKL mRNA decreased significantly in the HCC1 cells following RAL (10(-8), 10(-7)and 10(-6) M) treatment (% change from controls: 52+/-2*, 62+/-1*, 53+/-5.8*; *P<0.05). Similar results were seen in the HOB cells with RAL at 10(-6) M (RANKL mRNA: 72+/-5.5, P<0.05). In addition, there was a significant decrease in the RANKL/OPG ratio after RAL at 10(-6) M (HOB: 65.6+/-5*, HCC1: 56.9+/-20*; *P<0.05). RANKL/OPG ratio did not change significantly in the HCC1 cells following E(2). However, in contrast to RAL, we observed an increase in the RANKL/OPG ratio in the HOB cells following treatment with E(2). In conclusion, the study shows that RAL and E(2) have divergent cell-specific effects on the regulation of cytokines. The data also suggest that, in contrast to E(2), RAL may exert its anti-resorptive actions, at least in part, via the RANKL/OPG pathway. Further in vivo studies are required to confirm this.     

Induction of bone-type alkaline phosphatase in human vascular smooth muscle cells: roles of tumor necrosis factor-alpha and oncostatin M derived from macrophages

Inflammatory cells such as macrophages and T lymphocytes play an important role in vascular calcification associated with atherosclerosis and cardiac valvular disease. In particular, macrophages activated with cytokines derived from T lymphocytes such as interferon-gamma (IFN-gamma) may contribute to the development of vascular calcification. Moreover, we have shown the stimulatory effect of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) on in vitro calcification through increasing the expression of alkaline phosphatase (ALP), an ectoenzyme indispensable for bone mineralization, in vascular smooth muscle cells. Therefore, we hypothesized that macrophages may induce calcifying phenotype, especially the expression of ALP in human vascular smooth muscle cells (HVSMCs) in the presence of IFN-gamma and 1,25(OH)2D3. To test this hypothesis, we used cocultures of HVSMCs with human monocytic cell line (THP-1) or peripheral blood monocytes (PBMCs) in the presence of IFN-gamma and 1,25(OH)2D3. THP-1 cells or PBMCs induced ALP activity and its gene expression in HVSMCs and the cells with high expression of ALP calcified their extracellular matrix by the addition of beta-glycerophosphate. Thermostability and immunoassay showed that ALP induced in HVSMCs was bone-specific enzyme. We further identified tumor necrosis factor-alpha (TNF-alpha) and oncostatin M (OSM) as major factors inducing ALP in HVSMCs in the culture supernatants of THP-1 cells. TNF-alpha and OSM, only when applied together, increased ALP activities and in vitro calcification in HVSMCs in the presence of IFN-gamma and 1,25(OH)2D3. These results suggest that macrophages may contribute to the development of vascular calcification through producing various inflammatory mediators, especially TNF-alpha and OSM.     

Bone morphogenetic proteins regulate osteoprotegerin and its ligands in human vascular smooth muscle cells

The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved in the transformation of human vascular smooth muscle cells (HVSMC) to osteoblast-like cells. In this study, we evaluated the effect of BMP-2, BMP-7 and transforming growth factor beta (TGF-β1) on the secretion and mRNA expression of OPG and its ligands receptor activator of nuclear factor-κβ ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL) in HVSMC. All three growth factors decreased OPG protein production significantly; these results were paralleled by reduced OPG mRNA expression. TRAIL mRNA levels were also decreased. RANKL mRNA expression declined when treated with TGF-β1 but were increased by both BMPs. Members of the TGF-superfamily, i.e. TGF-β1, BMP-2 and BMP-7 exert effects on OPG and its ligands, indicating that these peptides may be involved in the development of vascular calcifications. The downregulation of OPG by these peptides does, however, not suggest that these factors are directly involved in OPG accumulation in diabetes.     

IGF-I regulates osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand in vitro and OPG in vivo

IGF-I, a ubiquitous polypeptide, plays a key role in longitudinal bone growth and acquisition. The most predominant effect of skeletal IGF-I is acceleration of the differentiation program for osteoblasts. However, in vivo studies using recombinant human (rh) IGF-I and/or rhGH have demonstrated stimulation of both bone formation and resorption, thereby potentially limiting the usefulness of these peptides in the treatment of osteoporosis. In this study, we hypothesized that IGF-I modulates bone resorption by regulating expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) in bone cells. Using Northern analysis in ST2 cells, we found that human IGF-I suppressed OPG mRNA in a time- and dose-dependent manner: 100 micro g/LIGF-I (13 nM) decreased OPG expression by 37.0 +/- 1.8% (P < 0.002). The half maximal inhibitory dose of IGF-I was reached at 50 micro g/liter ( approximately 6.5 nM) with no effect of IGF-I on OPG message stability. Conditioned media from ST2 cells confirmed that IGF-I decreased secreted OPG, reducing levels by 42%, from 12.1-7 ng/ml at 48 h (P < 0.05). Similarly, IGF-I at 100 micro g/liter (13 nM) increased RANKL mRNA expression to 353 +/- 74% above untreated cells as assessed by real-time PCR. In vivo, low doses of rhGH when administered to elderly postmenopausal women only modestly raised serum IGF-I (to concentrations of 18-26 nM) and did not affect circulating OPG concentrations; however, administration of rhIGF-I (30 micro g/kg.d) for 1 yr to older women resulted in a significant increase in serum IGF-I (to concentrations of 39-45 nM) and a 20% reduction in serum OPG (P < 0.05). In summary, we conclude that IGF-I in a dose- and time-dependent manner regulates OPG and RANKL in vitro and in vivo. These data suggest IGF-I may act as a coupling factor in bone remodeling by activating both bone formation and bone resorption; the latter effect appears to be mediated through the OPG/RANKL system in bone.     

T cells support osteoclastogenesis in an in vitro model derived from human multiple myeloma bone disease: the role of the OPG/TRAIL interaction

The development of multiple myeloma (MM) bone disease is mediated by increased number and activity of osteoclasts (OCs). Using an in vitro osteoclastogenesis model consisting of unstimulated and unfractionated peripheral blood mononuclear cells (PBMCs) from patients with MM, we showed that T cells support the formation of OCs with longer survival. Different from T-cell-depleted MM PBMC cultures, exogenous macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL) were necessary for the formation of OCs; however, they did not exhibit longer survival. We found up-regulated production of RANKL, osteoprotegerin (OPG), and TNF-related apoptosis-inducing ligand (TRAIL) by fresh MM T cells. Despite high OPG levels, the persistence of osteoclastogenesis can be related to the formation of the OPG/TRAIL complex demonstrated by immunoprecipitation experiments and the addition of anti-TRAIL antibody which decreases OC formation. OCs overexpressed TRAIL decoy receptor DcR2 in the presence of MM T cells and death receptor DR4 in T-cell-depleted cultures. In addition, increased Bcl-2/Bax (B-cell lymphoma-2/Bcl2-associated protein X) ratio, following Bcl-2 up-regulation, was detected in OCs generated in the presence of T cells. Our results highlight that MM T cells support OC formation and survival, possibly involving OPG/TRAIL interaction and unbalanced OC expression of TRAIL death and decoy receptors.     

Stimulation of resorption in cultured mouse calvarial bones by thiazolidinediones

Dosage-dependent release of 45Ca was observed from prelabeled mouse calvarial bones after treatment with two thiazolidinediones, troglitazone and ciglitazone. Release of 45Ca by ciglitazone was decreased by the osteoclast inhibitors acetazolamide, calcitonin, 3-amino-1-hydroxypropylidene-1,1-bisphosphonate, and IL-4, but not affected by the peroxisome proliferator-activated receptor gamma antagonist, GW 9662, the mitotic inhibitor, hydroxyurea, or indomethacin. Enhanced expression of receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA and protein and decreased osteoprotegerin (OPG) mRNA and protein were noted after ciglitazone treatment of calvariae. Ciglitazone and RANKL each caused increased mRNA expression of osteoclast markers: calcitonin receptor, tartrate-resistant acid phosphatase, cathepsin K, matrix metalloproteinase-9, integrin beta3, and nuclear factor of activated T cells 2. OPG inhibited mRNA expression of RANKL stimulated by ciglitazone, mRNA expression of osteoclast markers stimulated by ciglitazone and RANKL, and 45Ca release stimulated by troglitazone and ciglitazone. Increased expression of IL-1alpha mRNA by ciglitazone was not linked to resorption stimulated by the thiazolidinedione. Ciglitazone did not increase adipogenic gene expression but enhanced osteocalcin mRNA in calvariae. In addition to exhibiting sensitivity to OPG, data indicate that stimulation of osteoclast differentiation and activity by thiazolidinediones may occur by a nonperoxisome proliferator-activated receptor gamma-dependent pathway that does not require cell proliferation, prostaglandins, or IL-1alpha but is characterized by an increased RANKL to OPG ratio.     

Estrogen receptor alpha, but not estrogen receptor beta, is involved in the regulation of the OPG/RANKL (osteoprotegerin/receptor activator of NF-kappa B ligand) ratio and serum interleukin-6 in male mice.

Estrogens are important for the male skeleton. Osteoprotegerin (OPG), receptor activator of NF-kappa B ligand (RANKL), interleukin-6 (IL-6), IL-1 and tumor necrosis factor alpha (TNFalpha) have been suggested to be involved in the skeletal effects of estrogen. We treated orchidectomized mice with estradiol for 2 weeks and observed a 143% increase in the trabecular bone mineral density of the distal metaphysis of femur that was associated with a decreased OPG/RANKL mRNA ratio in vertebral bone. A similar decreased OPG/RANKL ratio was also seen after estrogen treatment of ovariectomized female mice. The effect of estrogen receptor (ER) inactivation on the OPG/RANKL ratio was dissected by using intact male mice lacking ER alpha (ERKO), ER beta (BERKO) or both receptors (DERKO). The expression of OPG was increased in ERKO and DERKO but not in BERKO male mice, resulting in an increased OPG/RANKL ratio. Furthermore, serum levels of IL-6 and tartrate-resistant acid phosphatase 5b (TRAP 5b) were decreased in ERKO and DERKO, but not in BERKO male mice. These results demonstrate that ER alpha, but not ER beta, is involved in the regulation of the vertebral OPG/RANKL ratio, serum levels of IL-6 and TRAP 5b in male mice.     

The RANKL/OPG system is activated in inflammatory bowel disease and relates to the state of bone loss

BACKGROUND AND AIMS: A substantial proportion of patients with inflammatory bowel disease (IBD) develops osteopenia and osteoporosis in the course of disease. Recent data from a mouse model of colitis suggest that the receptor activator of nuclear factor kappa B (RANKL)/osteoprotegerin (OPG) system may be responsible for bone loss. METHODS: We investigated the activation state of the RANKL/OPG system and its association with bone loss in human IBD. Plasma levels of OPG and RANKL were correlated with bone mineral density and current IBD therapy. Colonic secretion of OPG and RANKL and cell types responsible for such secretion were determined. RESULTS: OPG plasma levels were elevated 2.4-fold in Crohn's disease (CD) and 1.9-fold in ulcerative colitis (UC) whereas soluble RANKL (sRANKL) levels were not significantly different in IBD patients compared with healthy controls. High levels of OPG were released from colonic explant cultures (CEC) derived from inflamed IBD specimens, and colonic macrophages and dendritic cells costained for OPG. sRANKL levels from CEC were low both in IBD patients and healthy controls. Interestingly, increased expression of RANKL was mainly confined to cells in the lamina muscularis. A significant negative correlation was found between OPG plasma levels and femoral neck/lumbar spine bone mineral density. CONCLUSIONS: We have demonstrated that IBD is associated with alterations in the RANKL/OPG system. Applying results from a murine model of colitis associated bone loss, the constellation of OPG and sRANKL regulation observed in our study raises the possibility that RANKL/OPG may contribute to the development of bone loss in IBD.     

The differential expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) in human osteoarthritic subchondral bone osteoblasts is an indicator of the metabolic state of these disease cells

OBJECTIVE: We previously reported that human OA subchondral bone osteoblasts could be discriminated into two subpopulations identified by their levels of endogenous production (low [L] or high [H]) of PGE(2). Here, we investigated the OPG and RANKL expression levels, the histologic analysis of the subchondral bone as well as the osteoclast differentiation effect of osteoblasts on normal and both OA subpopulations (L and H), and further examined on the L OA osteoblasts the modulation of bone remodelling factors on the OPG and RANKL levels, as well as on the resorption activity. METHODS: Gene expression was determined using real-time PCR, PGE2 and OPG levels by specific ELISA, and membranous RANKL by flow cytometry. Histological observation of the subchondral bone was performed on human knee specimens. Osteoclast differentiation and formation was assayed by using the pre-osteoclastic cell line RAW 264.7. OPG and RANKL modulation on L OA osteoblasts was monitored following treatment with osteotropic factors, and the resorption activity was studied by the co-culture of differentiated PBMC/osteoblasts. RESULTS: Human OA subchondral bone osteoblasts expressed less OPG than normal. Compared to normal, RANKL gene expression levels were increased in L OA and decreased in H OA cells. The OPG/RANKL mRNA ratio was significantly diminished in L OA compared to normal or H OA (p<0.02, p<0.03), and markedly increased in H OA compared to normal. Inhibition of endogenous PGE(2) levels by indomethacin markedly decreased the ratio of OPG/RANKL on the H OA. In contrast to H OA osteoblasts, L OA cells induced a significantly higher level of osteoclast differentiation and formation (p<0.05).Histological analysis showed a reduced subchondral bone on the L OA and an increased bone mass on the H OA compared to normal. Treatment of L OA osteoblasts with osteotropic factors revealed that the OPG/RANKL mRNA expression ratio was significantly reduced by vitamin D(3) and significantly increased by TNF-alpha, PTH and PGE(2), while IL-1Beta demonstrated no effect. OPG protein levels showed similar profiles. No true effect was noted on membranous RANKL upon treatment with IL-1Beta, PGE(2) and PTH, but a significant increase was observed with vitamin D3 and TNF-alpha. The resorption activity of the L OA cells was significantly inhibited by all treatments except IL-1Beta, with maximum effect observed with vitamin D(3) and PGE(2). CONCLUSION: OPG and RANKL levels, and consequently the OPG/RANKL ratio, differed according to human OA subchondral bone osteoblast classification; it is decreased in L and increased in H OA. These findings, in addition to those showing that L OA osteoblasts have a reduced subchondral bone mass and induce a higher level of osteoclast differentiation, strongly suggest that the metabolic state of the L OA osteoblasts favours bone resorption.