Effects of the administration of corticosterone,parathyroid hormone,or both,and of their withdrawal,on rat bone and cartilage histomorphometric parameters,and on osteoprotegerin and RANKL mRNA expression and proteins

We have studied the effects of the treatment with corticosterone (CORT), parathyroid hormone (PTH), or both (CORT + PTH), and of their withdrawal (CORT-rec and CORT + PTH-rec), on the osteoprotegerin (OPG) and receptor activator of nuclear factor-kB ligand (RANKL) localization and expression and on histomorphometric parameters in primary and secondary spongiosa of rat femur and tibia metaphyses. In the secondary spongiosa of the CORT group, the bone remodeling and the OPG/RANKL mRNA ratio decreased. In the PTH group, the bone turnover and the structural and connectivity indices increased, and the OPG/RANKL mRNA ratio fell; this ratio rose, however, in the primary spongiosa. In the CORT + PTH group, remodeling values intermediate between those of the CORT and PTH groups, were detected in the secondary spongiosa, where OPG and RANKL mRNA rose. Return towards control values was found in the recovery groups. The Cartilage Growth Plate Width was reduced in the CORT and CORT + PTH groups and returned to normal values in the recovery groups, while it was not affected by PTH. Independently of treatments, both OPG and RANKL mRNA and proteins were co-localized in the same cartilage and bone cells and in several bone marrow cells. In conclusion, the catabolic effects induced by CORT treatment occur together with an OPG fall and a RANKL rise. In the PTH group in which the bone turnover increase, the OPG and RANKL mRNA expressions differ in the primary and secondary spongiosa, confirming that the bone tissue in these sites can have different metabolic trends.     

Effects of intermittent parathyroid hormone (PTH) administration on SOST mRNA and protein in rat bone

Summary Sclerostin, the secreted protein product of the SOST gene, which is mainly expressed by osteocytes, has recently been proposed as a negative regulator of bone osteoblastogenesis. Chronic elevation of PTH reduces SOST expression by osteocytes, while controversial results have been obtained by intermittent PTH administration. We have investigated the effects of intermittently administered PTH on SOST expression and sclerostin localization, comparing them with those of controls, as they appeared in three different bone segments of rat tibia: secondary trabecular metaphyseal and epiphyseal bone, and cortical diaphyseal bone. The histomorphometric results demonstrate that PTH enhances bone turnover through anabolic effects, as shown by the association of increased bone resorption variables with a significant rise in BV/TV, Tb.Th and Tb.N and a fall in Tb.Sp. PTH induces a SOST mRNA and protein fall in secondary metaphyseal trabeculae, diaphyseal bone and in epiphyseal trabeculae. Numbers of sclerostin immunopositive osteocytes/mm² show no change, compared with controls; there are fewer sclerostin-positive osteocytes in secondary metaphyseal trabeculae than in the other two bone areas, both in the control and PTH groups. The low numbers of sclerostin-positive osteocytes in the metaphyseal trabecular bone seem to be directly related to the fact that this area displays a high remodeling rate. The anabolic effects of PTH are in line with the fall of SOST mRNA and protein in all the three bone segments examined; the rise of bone turnover supports a negative role of SOST in bone formation.     

Detection of osteoprotegerin (OPG) and its ligand (RANKL) mRNA and protein in femur and tibia of the rat

Osteoprotegerin (OPG) and the receptor activator of nuclear factor (NF)-kB ligand (RANKL) are key regulators of osteoclastogenesis. The present study had the main aim of showing the localization of OPG and RANKL mRNA and protein in serial sections of the rat femurs and tibiae by immunohistochemistry (IHC) and in situ hybridization (ISH). The main results were: (1) OPG and RANKL mRNA and protein were co-localized in the same cell types, (2) maturative/hypertrophic chondrocytes, osteoblasts, lining cells, periosteal cells and early osteocytes were stained by both IHC and ISH, (3) OPG and RANKL proteins were mainly located in Golgi areas, and the ISH reaction was especially visible in active osteoblasts, (4) immunolabeling was often concentrated into cytoplasmic vacuoles of otherwise negative proliferative chondrocytes; IHC and ISH labeling increased from proliferative to maturative/hypertrophic chondrocytes, (5) the newly laid down bone matrix, cartilage-bone interfaces, cement lines, and trabecular borders showed light OPG and RANKL immunolabeling, (6) about 70% of secondary metaphyseal bone osteocytes showed OPG and RANKL protein expression; most of them were ISH-negative, (7) osteoclasts were mostly unstained by IHC and variably labeled by ISH. The co-expression of OPG and RANKL in the same bone cell types confirms their strictly coupled action in the regulation of bone metabolism.     

SOST基因的表达调控

硬化蛋白(Sclerostin, SOST)主要由骨细胞特异性表达, 是骨形成的负性调节因子。甲状旁腺激素和雌激素抑制SOST基因表达, 转录因子Osterix、Runx2和Mef2c促进SOST基因表达, 而转录因子Sirt1负调控SOST表达。此外, SOST基因表达还受DNA甲基化和microRNA等表观遗传学调控。SOST基因突变可引起骨硬缩症和Van Buchem病, 与骨质疏松症相关联。Wnt和BMP是骨代谢调节的两个重要信号途径, SOST可通过结合BMP的Ⅰ型或Ⅱ型受体和Wnt的共受体LRP5/6分别抑制BMP和Wnt信号途径来调控成骨细胞分化和骨形成。抑制SOST为骨质疏松症的治疗提供了新的途径。文章综述了SOST基因的结构、功能、表达调控、与人类疾病的关系、调节骨代谢的机制及其临床应用前景。     

Exendin-4 exerts osteogenic actions in insulin-resistant and type 2 diabetic states

Poor control of glucose homeostasis accounts for diabetes-related bone loss. Incretins – GLP-1 and GIP – have been proposed to affect bone turnover. GLP-1, apart from its anti-diabetic and other actions, has shown to exert a bone anabolic effect in streptozotocin-induced type 2 diabetic (T2D) and fructose-induced insulin-resistant (IR) rats. Exendin-4 (Ex-4), a peptide of non-mammalian nature, is sharing with GLP-1 part of its structural sequence, and also several glucoregulatory effects in mammals in an even more efficient manner. We have explored the effect of continuous administration (3 days by osmotic pump) of Ex-4 or saline (control) on bone turnover factors and bone structure in T2D and IR rats, compared to N, and the possible interaction of Ex-4 with the Wnt signalling pathway. Blood was taken before and after treatment for plasma measurements; tibiae and femurs were collected for gene expression of bone markers (RT-PCR) and structure (µCT) analysis; we also measured the mRNA levels of LRP5 – an activator of the Wnt pathway – and those of DKK1 and sclerostin (SOST) — both blockers of LRP5 activity. Compared to N-control, plasma glucose and insulin were respectively higher and lower in T2D; osteocalcin (OC) and tartrate-resistant alkaline phosphatase 5b (TRAP5b) were lower; after Ex-4, these turnover markers were further reduced in T2D and IR, while TRAP5b increased in N. Bone OC, osteoprogeterin (OPG) and receptor activator of NF-kB ligand (RANKL) mRNA were lower in T2D and IR; Ex-4 increased OC in all groups and OPG in N and IR, reduced RANKL in N and T2D but increased it in IR; the LRP5/DKK1 and LRP5/SOST mRNA ratios were similarly decreased in T2D, but in IR, the latter ratio was reduced while the former was increased; after Ex-4, both ratios augmented in N, and that of LRP5/DKK1 tended to normalize in T2D and IR. In conclusion, Ex-4 exerts osteogenis effects in T2D and IR models, and interacts with the Wnt pathway to promote bone formation.     

Mediators of the biphasic responses of bone to intermittent and continuously administered parathyroid hormone

Parathyroid hormone (PTH) has biphasic effects on bone: continuous treatment is catabolic whereas intermittent treatment is anabolic. The mechanism(s) responsible for these differing effects are still unclear, partly because of the previous non-availability of a model system in which effects on both formation and resorption indices could be studied concomitantly. In cultured marrow cells from 6-week old C57BL/6 mice, we demonstrated that 4 days of intermittent PTH treatment increased mRNA for osteoblast differentiation markers (Runx2, alkaline phosphatase (AP), and type I procollagen (COL1A1) whereas continuous treatment resulted in production of large numbers of TRAP-positive multinucleated osteoclasts. Although IGF-I mRNA did not increase after intermittent treatment, it was consistently higher than after continuous treatment, and the addition of an anti-IGF-I neutralizing antibody prevented the increase in bone formation indices observed with intermittent treatment. By contrast, after continuous treatment, gene expression of RANK ligand (RANKL) was increased and that of osteoprotegerin (OPG) was decreased, resulting in a 25-fold increase in the RANKL/OPG ratio. In this model system, the data suggest that intermittent PTH treatment enhances osteoblast differentiation through an IGF-I dependent mechanism and continuous PTH treatment enhances osteoclastogenesis through reciprocal increases in RANKL and decreases in OPG.     

In vivo administration of 1,25-dihydroxyvitamin D3 suppresses the expression of RANKL mRNA in bone of thyroparathyroidectomized rats constantly infused with PTH

It is known that pharmacological or toxic doses of vitamin D induce bone resorption both in vivo and in vitro, whereas physiological doses of the vitamin have a protective effect on bone in vivo. To investigate the discrepancies of the dose-dependent effect of vitamin D on bone resorption, we examined the in vivo effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] on the expression of the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) and osteoprotegerin (OPG) mRNAs in bone of thyroparathyroidectomized (TPTX) rats infused with or without parathyroid hormone (PTH). Continuous infusion of 50 ng/h of PTH greatly increased the expression of RANKL mRNA in bone of TPTX rats. Expression of OPG mRNA was not altered by PTH infusion. When graded doses of 1,25(OH)(2)D(3) was daily administered orally for 14 days to normocalcemic TPTX rats constantly infused with PTH, 0.01 and 0.1 microg/kg of 1,25(OH)(2)D(3) inhibited the PTH-induced RANKL mRNA expression, but 0.5 microg/kg of the vitamin did not inhibit it. Regulator of G protein signaling-2 (RGS-2) gene expression was suppressed by 1,25(OH)(2)D(3) dose-dependently, but PTH/PTHrP receptor mRNA expression was not altered. Bone morphometric analyses revealed that 1,25(OH)(2)D(3) suppressed PTH-induced osteoclast number in vivo. These results suggest that pharmacological or toxic doses of 1,25(OH)(2)D(3) stimulate bone resorption by inducing RANKL, but a certain range of physiological doses of the vitamin inhibit PTH-induced bone resorption, the latter mechanism appeared to be mediated, at least in part, by the suppression of the PTH/PTHrP receptor-mediated signaling.     

SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor

Sclerosteosis is an autosomal recessive disease that is characterized by overgrowth of bone tissue and is linked to mutations in the gene encoding the secreted protein SOST. Sclerosteosis shares remarkable similarities with "high bone mass" diseases caused by "gain-of-function" mutations in the LRP5 gene, which encodes a coreceptor for Wnt signaling proteins. We show here that SOST antagonizes Wnt signaling in Xenopus embryos and mammalian cells by binding to the extracellular domain of the Wnt coreceptors LRP5 and LRP6 and disrupting Wnt-induced Frizzled-LRP complex formation. Our findings suggest that SOST is an antagonist for Wnt signaling and that the loss of SOST function likely leads to the hyperactivation of Wnt signaling that underlies bone overgrowth seen in sclerosteosis patients.     

Effects of arachidonic acid, docosahexaenoic acid, prostaglandin E(2) and parathyroid hormone on osteoprotegerin and RANKL secretion by MC3T3-E1 osteoblast-like cells

Bone is continuously remodeled through resorption by osteoclasts and the subsequent synthesis of the bone matrix by osteoblasts. Cell-to-cell contact between osteoblasts and osteoclast precursors is required for osteoclast formation. RANKL (receptor activator of nuclear factor-kappaB ligand) expressed on osteoblastic cell membranes stimulates osteoclastogenesis, while osteoprotegerin (OPG) secreted by osteoblasts inhibits osteoclastogenesis. Although polyunsaturated fatty acids (PUFAs) have been implicated in bone homeostasis, the effects thereof on OPG and RANKL secretion have not been investigated. MC3T3-E1 osteoblasts were exposed to the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA); furthermore, the bone-active hormone parathyroid hormone (PTH) and the effects thereof were tested on OPG and RANKL secretion. Prostaglandin E(2) (PGE(2)), a product of AA metabolism that was previously implicated in bone homeostasis, was included in the study. AA (5.0-20 microg/ml) inhibited OPG secretion by 25-30%, which was attenuated by pretreatment with the cyclooxygenase blocker indomethacin, suggesting that the inhibitory effect of AA on OPG could possibly be PGE(2)-mediated. MC3T3-E1 cells secreted very low basal levels of RANKL, but AA stimulated RANKL secretion, thereby decreasing the OPG/RANKL ratio. DHA suppressed OPG secretion to a smaller extent than AA. This could, however, be due to endogenous PGE(2) production. No RANKL could be detected after exposing the MC3T3-E1 cells to DHA. PTH did not affect OPG secretion, but stimulated RANKL secretion. This study demonstrates that AA and PTH reduce the OPG/RANKL ratio and may increase osteoclastogenesis. DHA, however, had no significant effect on OPG or RANKL in this model.     

Canonical Wnt signaling is required for Panax notoginseng saponin-mediated attenuation of the RANKL/OPG ratio in bone marrow stromal cells during osteogenic differentiation

Panax notoginseng saponins (PNS) are known to regulate the osteogenic differentiation of bone marrow stromal cells (BMSCs). In the present study, we investigated whether PNS could promote the osteogenic differentiation of BMSCs through modulating the Wnt/β-catenin signaling pathways, which are implicated in BMSCs osteogenesis. We found that PNS enhanced the mRNA expression of OPG, β-catenin, and cyclin D1 while decreased the mRNA expression of RANKL and PPARγ2. The actions of PNS on BMSCs were reversed (or partially) by DKK-1, a classical inhibitor of Wnt/β-catenin signaling. These results suggest that PNS stimulating bone formation by promoting the proliferation and osteogenic differentiation of BMSCs, and could also protect the skeletal system by decreasing bone resorption through reduction of RANKL/OPG expression via Wnt/β-catenin signaling pathways.     

IL-6 is not required for parathyroid hormone stimulation of RANKL expression, osteoclast formation, and bone loss in mice

Continuous elevation of parathyroid hormone (PTH) increases osteoclast precursors, the number of osteoclasts on cancellous bone, and bone turnover. The essential molecular mediators of these effects are controversial, however, and both increased receptor activator of NF-kappaB ligand (RANKL) and IL-6 have been implicated. The goal of these studies was to determine whether continuous elevation of endogenous PTH alters IL-6 gene expression in vivo and whether IL-6 is required for PTH-induced bone loss. To accomplish this, we generated transgenic mice harboring a luciferase reporter gene under the control of IL-6 gene regulatory regions to allow accurate quantification of IL-6 gene activity in vivo. In these mice, induction of secondary hyperparathyroidism using a calcium-deficient diet did not alter IL-6-luciferase transgene expression, whereas RANKL mRNA expression was elevated in bone tissue. Moreover, secondary hyperparathyroidism induced an equivalent amount of bone loss in wild-type and IL-6-deficient mice, and PTH elevated RANKL mRNA and osteoclast formation to the same extent in bone marrow cultures derived from wild-type and IL-6-deficient mice. These results demonstrate that IL-6 is not required for the osteoclast formation and bone loss that accompanies continuous elevation of PTH.     

Secretion of MUC5AC mucin from pancreatic cancer cells in response to forskolin and VIP

Bisphosphonates are potent antiresorptive drugs commonly employed in the treatment of metabolic bone diseases. Despite their frequent use, the mechanisms of bisphosphonates on bone cells have largely remained unclear. Receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for osteoclast formation and activation, whereas osteoprotegerin (OPG) neutralizes RANKL. Various osteotropic drugs have been demonstrated to modulate osteoblastic production of RANKL and OPG. In this study, we assessed the effects of the bisphosphonates pamidronate (PAM) and zoledronic acid (ZOL) on OPG mRNA steady-state levels (by semiquantitative RT-PCR) and protein production (by ELISA) in primary human osteoblasts (hOB). PAM increased OPG mRNA levels and protein secretion by hOB by up to 2- to 3-fold in a dose-dependent fashion with a maximum effect at 10(-6) M (P < 0.001) after 72 h. Similarly, ZOL enhanced OPG gene expression and protein secretion by hOB in a dose-dependent fashion with a maximum effect at 10(-8) M after 72 h, consistent with the higher biological potency of ZOL. Time course experiments indicated a stimulatory effect of PAM and ZOL on osteoblastic OPG protein secretion by 6-fold, respectively (P < 0.001). Pretreatment with PAM and ZOL prevented the inhibitory effects of the glucocorticoid dexamethasone on OPG mRNA and protein production. Analysis of cellular markers of osteoblastic differentiation revealed that PAM and ZOL induced type I collagen secretion and alkaline phosphatase activity by 2- and 4-fold, respectively (P < 0.0001 by ANOVA). In conclusion, our data suggest that bisphosphonates modulate OPG production by normal human osteoblasts, which may contribute to the inhibition of osteoclastic bone resorption. Since, OPG production increases with osteoblastic cell maturation, enhancement of OPG by bisphosphonates could be related to their stimulatory effects on osteoblastic differentiation.     

Supplementation with Fish Oil and Genistein,Individually or in Combination,Protects Bone against the Adverse Effects of Methotrexate Chemotherapy in Rats

Cancer chemotherapy has been shown to induce long-term skeletal side effects such as osteoporosis and fractures; however, there are no preventative treatments. This study investigated the damaging effects of anti-metabolite methotrexate (MTX) subcutaneous injections (0.75 mg/kg BW) for five days and the potential protective benefits of daily oral gavage of fish oil at 0.5 mL/100 g BW (containing 375 mg of n-3 PUFA/100 g BW), genistein (2 mg/100 g BW), or their combination in young adult rats. MTX treatment alone significantly reduced primary spongiosa height and secondary spongiosa trabecular bone volume. Bone marrow stromal cells from the treated rats showed a significant reduction in osteogenic differentiation but an increase in adipogenesis ex vivo. Consistently, stromal cells had significantly higher mRNA levels of adipogenesis-related proliferator activator activated receptor-γ (PPAR-γ) and fatty acid binding protein (FABP4). MTX significantly increased the numbers of bone-resorbing osteoclasts and marrow osteoclast precursor cell pool while significantly enhancing the mRNA expression of receptor activator for nuclear factor kappa B ligand (RANKL), the RANKL/osteoprotegerin (OPG) ratio, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the bone. Supplementary treatment with fish oil and/or genistein significantly preserved trabecular bone volume and osteogenesis but suppressed MTX-induced adipogenesis and increases in osteoclast numbers and pro-osteoclastogenic cytokine expression. Thus, Fish oil and/or genistein supplementation during MTX treatment enabled not only preservation of osteogenic differentiation, osteoblast number and bone volume, but also prevention of MTX treatment-induced increases in bone marrow adiposity, osteoclastogenic cytokine expression and osteoclast formation, and thus bone loss.     

Receptor activator of NF-kappaB ligand protein expression in UMR-106 cells is differentially regulated by parathyroid hormone and calcitriol

Expression of the cytokine, receptor activator of NF-kappaB ligand (RANKL), is stimulated by both parathyroid hormone (PTH) and calcitriol in osteoblasts. Most studies have examined the effects on RANKL mRNA, and less information is available on the protein products. We have determined the effects of PTH, the adenylate cyclase stimulator forskolin, and calcitriol, alone and in combination, on endogenous RANKL protein expression in UMR-106 rat osteoblastic osteosarcoma cells by Western blotting and enzyme immunoassay (EIA). PTH and forskolin dose dependently increased a approximately 52 kDa band in whole cell lysates that was detected by both C- and N-terminal directed RANKL antibodies. Calcitriol treatment produced little or no expression of this approximately 52 kDa band, but markedly increased the expression of a approximately 32 kDa band that was only detected with an antibody directed to the N-terminus of RANKL. An EIA based on RANKL binding to OPG detected a large increase in RANKL expression following calcitriol treatment, and much smaller increases with PTH or forskolin. The combination of PTH and calcitriol or forskolin and calcitriol elicited effects similar to those of PTH and forskolin alone, as detected by both Western blotting and EIA. In contrast to the effects on protein, all agents increased RANKL mRNA expression, with the greatest effects seen with the co-treatments. The results indicate that PTH, likely through effects on cyclic AMP, has a different effect on RANKL processing than calcitriol. The approximately 52 and approximately 32 kDa RANKL products appear to interact differently with OPG, which could affect responses to the agents in target cells.     

Effects of parathyroid hormone on Wnt signaling pathway in bone

The Wnt signaling pathway has recently been demonstrated to play an important role in bone cell function. In previous studies using DNA microarray analyses, we observed a change in some of the molecular components of the canonical Wnt pathway namely, frizzled-1 (FZD-1) and axil, in response to continuous parathyroid hormone (PTH) treatment in rats. In the present study, we further explored other components of the Wnt signaling pathway in rat distal metaphyseal bone in vivo, and rat osteoblastic osteosarcoma cells (UMR 106) in culture. Several Wnt pathway components, including low-density lipoprotein-receptor-related protein 5 (LRP5), LRP6, FZD-1, Dickkopf-1 (Dkk-1), and Kremen-1 (KRM-1), were expressed in bone in vivo and in osteoblasts in vitro. Continuous exposure to PTH (1-38) both in vivo and in vitro upregulated the mRNA expression of LRP6 and FZD-1 and decreased LRP5 and Dkk-1. These effects in UMR 106 cells were associated with an increase in beta-catenin as measured by Western blots and resulted in functional activation (three to six-fold) of a downstream Wnt responsive TBE6-luciferase (TCF/LEF-binding element) reporter gene. Activation of the TBE6-luciferase reporter gene by PTH (1-38) in UMR 106 cells was inhibited by the protein kinase A (PKA) inhibitor, H89. Activation was mimicked by PTH (1-31), PTH-related protein (1-34), and forskolin, but both PTH (3-34) and (7-34) had no effect. These findings suggest that the effect of PTH on the canonical Wnt signaling pathway occurs at least in part via the cAMP-PKA pathway through the differential regulation of the receptor complex proteins (FZD-1/LRP5 or LRP6) and the antagonist (Dkk-1). Taken together, these results reveal a possible role for the Wnt signaling pathway in PTH actions in bone.     

Leptin effect on RANKL and OPG expression in MC3T3-E1 osteoblasts

Recent studies have suggested that leptin hormone may play a pivotal role on bone remodeling through a direct effect by modulating positively the OPG/RANKL balance. Here, we investigate the effect of leptin hormone on RANKL and OPG expression in MC3T3-E1 osteoblasts using RT-PCR and ELISA measurements. We have at first identified the expression of Ob-Rb and Ob-Ra leptin receptor isoforms in MC3T3-E1 and observed that these cells respond to mrleptin treatments. We then investigated the effect of mrleptin on RANKL and OPG expression. We show that mrleptin dose-dependently regulated the expression of RANKL mRNA with complete inhibition observed at concentrations higher than 12 ng/ml. This effect was confirmed with sRANKL protein measurements. However, the exposure of MC3T3-E1 to mrleptin had no effect on OPG mRNA. Taken together, these results suggest that leptin modulates positively OPG/RANKL balance by inhibiting the expression of RANKL gene.     

Detection and characterization of RANK ligand and osteoprotegerin in the thyroid gland

Receptor activator of NF-kappaB (RANK) ligand (RANKL) and osteoprotegerin (OPG) play essential roles in bone metabolism and immune responses. RANKL activates RANK, which is expressed by osteoclasts and dendritic cells (DC), whereas OPG acts as its decoy receptor. The role of RANKL and OPG in thyroid physiology is unclear. Northern analysis revealed pronounced OPG mRNA levels in normal human thyroid. By contrast, RANKL mRNA levels were most abundant in lymph node and appendix, and low in the thyroid. In the human thyroid follicular cell line XTC and in primary human thyroid follicular cells, OPG mRNA levels and protein secretion were upregulated by interleukin (IL)-1beta (33-fold), tumor necrosis factor (TNF)-alpha (eightfold), and thyrotropin (TSH) (threefold). RANKL mRNA was stimulated in XTC by IL-1beta and TNF-alpha, but inhibited by TSH. Conditioned medium harvested from IL-1beta-treated XTC (containing high concentrations of OPG) inhibited RANKL-induced CD40 upregulation and cluster formation of DC. OPG mRNA levels were three times more abundant in surgical thyroid specimens of Graves' disease as compared to other thyroid diseases. Our data suggest that RANKL and OPG are produced in the thyroid gland by thyroid follicular cells, are regulated by cytokines and TSH, and are capable of modulating dendritic cell functions. Thus, these cytokines may represent important local immunoregulatory factors involved in the pathogenesis of autoimmune thyroid diseases.