Cytokine-induced inhibition of bone matrix proteins is not mediated by prostaglandins

Interleukin-1 (IL-1) and tumor necrosis factor (TNF), two pleiotropic cytokines produced in inflammatory processes, inhibit bone matrix biosynthesis and stimulate prostanoid formation in osteoblasts. In the present study, the importance of prostaglandin formation in IL-1 and TNF-induced inhibition of osteocalcin and type I collagen formation has been examined. In the human osteoblastic cell line MG-63, IL-1 alpha (10-1000 pg/ml), IL-1 beta (3-300 pg/ml) and TNF-alpha (1-30 ng/ml) stimulated prostaglandin E2 (PGE2) formation and inhibited 1,25(OH)2-vitamin D3-induced osteocalcin biosynthesis as well as basal production of type I collagen. Addition of PGE2 or increasing the endogenous formation of PGE2 by treating the cells with arachidonic acid, bradykinin, Lys-bradykinin or des-Arg9-bradykinin, did not affect osteocalcin and type I collagen formation in unstimulated or 1,25(OH)2-vitamin D3-stimulated osteoblasts. Four non-steroidal antiinflammatory drugs, indomethacin, flurbiprofen, naproxen and meclofenamic acid, inhibited basal, IL-1 beta- and TNF-alpha-stimulated PGE2 formation in the MG-63 cells without affecting IL-1 beta- or TNF-alpha-induced inhibition of osteocalcin and type I collagen formation. In isolated, non-transformed, human osteoblast-like cells, IL-1 beta and TNF-alpha stimulated PGE2 formation and concomitantly inhibited 1,25(OH)2-vitamin D3-stimulated osteocalcin biosynthesis, without affecting type I collagen formation. In these cells, indomethacin and flurbiprofen abolished the effects of IL-1 beta and TNF-alpha on prostaglandin formation without affecting the inhibitory effects of the cytokines on osteocalcin biosynthesis. These data show that IL-1 and TNF inhibit osteocalcin and type I collagen formation in osteoblasts independently of prostaglandin biosynthesis and that non-steroidal antiinflammatory drugs do not affect the effects of IL-1 and TNF on bone matrix biosynthesis.     

Recombinant human bone morphogenetic protein-2 stimulates osteoblast differentiation and suppresses matrix metalloproteinase-1 production in human bone cells isolated from mandibulae

Bone morphogenetic protein (BMP), a member of the transforming growth factor superfamily, is one of the most potent growth factors that stimulate osteoblast differentiation and bone formation. We investigated the effects of recombinant human BMP-2 (rhBMP-2) on osteoblast differentiation and matrix metalloproteinase-1 (MMP-1) production in human bone cells (HBC) isolated from mandibulae of 3 adult patients. rhBMP-2 at concentrations over 50 ng/ml significantly stimulated alkaline phosphatase activity and parathyroid hormone (PTH)-dependent 3', 5'-cyclic adenosine monophosphate accumulation, which are early markers of osteoblast differentiation, in HBCs. rhBMP-2 (500 ng/ml) also enhanced the level of PTH/PTH related-peptide receptor mRNA expression in HBCs. Although neither HBCs untreated nor treated with rhBMP-2 produced measurable amounts of osteocalcin, which is a marker of more mature osteoblasts, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced ostocalcin mRNA expression and its protein synthesis in these cells. rhBMP-2 inhibited 1,25(OH)2D3-induced osteocalcin synthesis in HBCs at both the mRNA and protein level. rhBMP-2 also significantly suppressed MMP-1 production and MMP-1 mRNA expression at concentrations over 500 ng/ml. These results suggest that rhBMP-2 exerts anabolic effects on human osteoblastic cells derived from mandibulae by stimulation of osteoblast differentiation and down-regulation of MMP-1 synthesis.     

Hypocalcemic effect of osteoclastogenesis inhibitory factor/osteoprotegerin in the thyroparathyroidectomized rat

Osteoclastogenesis inhibitory factor (OCIF), also termed as osteoprotegerin (OPG), is a soluble member of the tumor necrosis factor receptor family. Although OCIF/OPG is shown to inhibit osteoclast formation in vitro and prevent ovariectomy-induced bone loss in vivo, its effect on serum calcium level remains to be determined. In this study we examined the acute effect of OCIF on thyroparathyroidectomized rats whose serum calcium concentrations were raised either by exogenous PTH or 1,25-(OH)2D3. When OCIF was administered at the start of PTH infusion, it attenuated the initial rise in serum calcium. When OCIF was administered into rats with established hypercalcemia, it decreased serum calcium rapidly (within 2 hr) and dramatically. OCIF did not increase urinary calcium excretion. These findings, especially the rapid onset of its hypocalcemic effect, suggest that OCIF not only inhibits the formation of osteoclasts but also affects the function and/or survival of mature osteoclasts at doses used in this study.     

Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL

Osteoclasts, the multinucleated cells that resorb bone, develop from hematopoietic cells of monocyte/macrophage lineage. Osteoclast-like cells (OCLs) are formed by coculturing spleen cells with osteoblasts or bone marrow stromal cells in the presence of bone-resorbing factors. The cell-to-cell interaction between osteoblasts/stromal cells and osteoclast progenitors is essential for OCL formation. Recently, we purified and molecularly cloned osteoclastogenesis-inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF is a secreted member of the tumor necrosis factor receptor family and inhibits osteoclastogenesis by interrupting the cell-to-cell interaction. Here we report the expression cloning of a ligand for OPG/OCIF from a complementary DNA library of mouse stromal cells. The protein was found to be a member of the membrane-associated tumor necrosis factor ligand family and induced OCL formation from osteoclast progenitors. A genetically engineered soluble form containing the extracellular domain of the protein induced OCL formation from spleen cells in the absence of osteoblasts/stromal cells. OPG/OCIF abolished the OCL formation induced by the protein. Expression of its gene in osteoblasts/stromal cells was up-regulated by bone-resorbing factors. We conclude that the membrane-bound protein is osteoclast differentiation factor (ODF), a long-sought ligand mediating an essential signal to osteoclast progenitors for their differentiation into osteoclasts. ODF was found to be identical to TRANCE/RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only osteoclastogenesis but also immune system.     

Increase of vascular endothelial growth factor mRNA expression by 1,25-dihydroxyvitamin D3 in human osteoblast-like cells

Vascular endothelial growth factor (VEGF), a secreted endothelial cell-specific mitogen, is produced in endocrine organs and regulated by trophic hormones. Because angiogenesis and osteogenesis are closely regulated, we studied whether human osteoblast-like cells produce VEGF, and if so, what factors regulate VEGF mRNA expression. Human osteoblast-like cells (HObLC) derived from trabecular bone explants were cultured in alpha-MEM supplemented with 10% fetal calf serum. Northern blot analysis revealed that HObLC expressed VEGF mRNA, as did several human osteosarcoma cells. 1,25-(OH)2D3 increased the steady-state levels of VEGF mRNA in a time- and concentration-dependent manner in HObLC and one of the osteosarcoma cell lines, SaOS-2, accompanied by an increase in the concentration of immunoreactive VEGF in the conditioned medium. PTH and IGF-I also increased the level of VEGF mRNA in HObLC and SaOS-2 cells. Furthermore, 12-O-tetradecanoylphorbol ester stimulated VEGF mRNA in a time-and concentration-dependent manner. The VEGF mRNA expression induced by 1,25-(OH)2D3 was completely inhibited by H-7, but only partially by staurosporine. We have demonstrated that PTH, IGF-I, and most potently 1,25-(OH)2D3 stimulate the mRNA expression and secretion of VEGF in human osteoblast-like cells, suggesting that one of the anabolic effects of 1,25-(OH)2D3 on skeletal tissue may be mediated by VEGF produced by osteoblasts.     

Influence of aluminum on the regulation of PTH- and 1,25(OH)2D3-dependent pathways in the rat osteosarcoma cell line ROS 17/2.8

The role of hormonal status in the development of aluminum (Al)-dependent renal osteodystrophy, which is characterized by reduced bone matrix deposition, still remains largely unknown. To address this question, we used the osteoblast-like osteosarcoma cell line ROS 17/2.8 to evaluate the role of Al on parathyroid hormone (PTH)- and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent activities in these cells. Al (1 microM) caused an inhibition of basal and 1,25(OH)2D3-induced alkaline phosphatase, but only at low doses (< 1 nM) of the steroid. Al partly inhibited basal osteocalcin (OC) secretion in ROS cells (p < 0.001), and the dose-dependent increase in 1,25(OH)2D3-induced OC release by these cells was also reduced by 1 microM Al at low concentrations of the steroid (< or = 1 nM), whereas high doses of 1,25(OH)2D3 (> or = 5 nM) totally prevented the inhibiting effects of Al. Al also had strong inhibitory actions on PTH-dependent cAMP production by ROS cells over the concentration range tested (0.5-50 nM). This inhibitory action of Al was also observed for PTH-related peptide- (PTHrp, 50 nM) but not for Isoproterenol-dependent (100 nM) cAMP formation. To evaluate more fully the mechanism of this inhibition of cAMP formation, we investigated the effect of Al on toxin-modulated, G protein-dependent regulation of cAMP formation and on the activation of adenylate cyclase by Forskolin. Cholera toxin (CT, 10 micrograms/ml), applied to cells for 4 h prior to PTH challenge, enhanced cAMP production about 2-fold above PTH alone (p < 0.001), a process that was further stimulated by Al. Pertussis toxin (PT, 1 microgram/ml, 4 h) did not modify basal PTH-dependent cAMP formation by ROS cells. However, PT treatment prevented the inhibitory effect of Al on cAMP formation by these cells (p < 0.025). The stimulation of adenylate cyclase by Forskolin (0.1 and 1 microM), which bypasses G protein regulation, was not modified by Al, indicating that Al does not affect adenylate cyclase directly. Northern blot analysis of PTH receptor mRNA levels showed that Al did not modify PTH receptor message in ROS cells. Likewise, Western blot analyses of G protein subunits showed that Al did not significantly alter Gs alpha subunit levels, in accordance with the results obtained for cAMP-dependent formation in response to CT. In contrast, Gi alpha-1 and Gi alpha-2 subunits were decreased by Al treatment, consistent with PT-restricted increases in cAMP formation in Al-treated ROS cells. Taken together, these results suggest that Al has multiple actions in osteoblast-like ROS cells. The effects of Al are modulated by hormonal control of the pathways investigated. Al affects 1,25(OH)2D3-regulated functions only when this steroid is low. Al has large inhibitory effects on PTH- and PTHrp-dependent cAMP formation. This last feature is related to the ability of Al to alter the G protein transducing pathway for PTH/PTHrp-dependent formation of cAMP since it does not affect adenylate cyclase activity directly and does not affect the PTH receptor message level. Thus, Al has stronger deleterious effects in osteoblast-like cells with an already compromised 1,25(OH)2D3 status and can modulate specifically PTH/PTHrp-mediated cAMP formation at the postreceptor level.     

In vivo regulation of chromogranin A messenger ribonucleic acid in the parathyroid by 1,25-dihydroxyvitamin D: studies in normal rats and in chronic renal insufficiency

Chromogranin-A (CgA) and PTH are the two major secretory products of the parathyroid gland. In vitro, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] increases CgA, but decreases PTH messenger RNA (mRNA) levels. We investigated the physiological significance of the induced changes in CgA expression by examining the effects of 1,25-(OH)2D3 on parathyroid CgA mRNA levels in vivo. Normal rats were injected with 1,25-(OH)2D3 at 48 and 24 h before blood sampling and isolation of both parathyroid glands. Parathyroid total RNA was extracted and CgA and PTH mRNA quantified by Northern blot analysis. CgA mRNA levels increased 1.6-, 3.2- and 5.6-fold, whereas PTH mRNA levels decreased by 37, 63 and 97%, respectively, with 1,25-(OH)2D3 doses of 10, 50, and 250 pmol/100 g BW. Parathyroid gland CgA expression also was examined in rats with mild chronic renal insufficiency, induced by a 5/6 nephrectomy 5 weeks earlier. Chronic renal insufficiency rats, fed normal chow, had elevated serum urea, creatinine, and PTH levels and reduced 1,25-(OH)2D3 but normal serum levels of calcium and phosphate. PTH mRNA levels were elevated 4-fold and CgA mRNA levels were 50% lower in the uremic animals. This indicates that the regulation of CgA expression in normocalcemic rats occurs at physiological 1,25-(OH)2D3 concentrations. In summary, increases and decreases in serum 1,25-(OH)2D3 levels are associated with corresponding increases and decreases in CgA mRNA levels in the parathyroid glands of rats. Therefore, this study is the first to demonstrate the physiological relevance of the earlier in vitro observations.     

Induction of cyclooxygenase-2 by parathyroid hormone in human osteoblasts in culture

OBJECTIVE: Parathyroid hormone (PTH) induced bone resorption by osteoclasts depends on the presence of osteoblasts. PTH induced production of prostaglandins by osteoblasts and induction of bone resorption by prostaglandins suggest that these autacoids may be implicated in the effects of PTH on bone. Our objective was to determine if the increase in prostaglandin production induced in human osteoblasts by PTH is due to an increase in cyclooxygenase-2 (COX-2) expression. METHODS: Primary cultures of human osteoblasts were obtained from specimens of trabecular bone. Confluent cells were treated with PTH, dexamethasone or compound NS-398, a specific COX-2 inhibitor. The concentration of prostaglandin E2 (PGE2) in the supernatants was determined by radioimmunoassay and COX-2 mRNA levels evaluated by Northern blot. RESULTS: PTH induced COX-2 mRNA expression and PGE2 production. These effects were time and concentration dependent and were inhibited by dexamethasone. Compound NS-398 reduced PGE2 production to the same extent as dexamethasone, and neither compound had an additive effect on this variable. CONCLUSION: These results show that PTH induces COX-2 expression in human osteoblasts in culture and suggest that this isoenzyme is the main factor in the control of prostaglandin synthesis in these experimental conditions.     

Stimulation of osteoclast formation by 1,25-dihydroxyvitamin D requires its binding to vitamin D receptor (VDR) in osteoblastic cells: studies using VDR knockout mice

Previous studies have shown that 1,25-dihydroxyvitamin D [1,25(OH)2D] plays important roles in the formation of osteoclasts through its actions on osteoblastic cells. We have generated mice lacking vitamin D receptor (VDR) by gene targeting (VDR-/-). These mice had tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and exhibited similar levels of parameters for bone resorption to those in wild type mice. The present studies were undertaken to clarify whether effects of 1,25(OH)2D on osteoclast formation require VDR in osteoblasts, and to examine mechanisms of the formation of osteoclasts without VDR-mediated actions using VDR-/- mice. When wild-type calvarial osteoblasts and spleen cells were co-cultured with 1,25(OH)2D, TRAP-positive osteoclasts were formed regardless of the genotypes of spleen cells. In contrast, when osteoblasts from VDR-/- mice were co-cultured, no osteoclasts could be formed even with wild-type spleen cells. Parathyroid hormone and interleukin-1alpha stimulated osteoclast formation by co-cultures from VDR-/- mice, and the generated osteoclasts showed resorbing activity. These results demonstrate that VDR-mediated actions of 1,25(OH)2D in osteoblasts are essential for osteoclast formation by 1,25(OH)2D, and that functionally intact osteoclasts can be formed without 1,25(OH)2D actions under stimulations by other agents. It is suggested that osteoclastic bone resorption can be maintained without 1,25(OH)2D actions by other stimulatory agents.     

1alpha,25-dehydroxyvitamin D3 synergism toward transforming growth factor-beta1-induced AP-1 transcriptional activity in mouse osteoblastic cells via its nuclear receptor

The present study demonstrates 1alpha,25-dehydroxyvitamin D3 (1alpha-25-(OH)2D3) synergism toward transforming growth factor (TGF)-beta1-induced activation protein-1 (AP-1) activity in mouse osteoblastic MC3T3-E1 cells via the nuclear receptor of the vitamin. 1alpha-25-(OH)2D3 synergistically stimulated TGF-beta1-induced expression of the c-jun gene in the cells but not that of the c-fos gene. We actually showed by a gel mobility shift assay 1alpha-25-(OH)2D3 synergism of TGF-beta1-induced AP-1 binding to the 12-(O-tetradecanoylphorbol-13-acetate response element (TRE). 1alpha-25-(OH)2D3 markedly stimulated the transient activity of TGF-beta1-induced AP-1 in the cells transfected with a TRE-chloramphenicol acetyltransferase (CAT) reporter gene. Also, a synergistic increase in TGF-beta1-induced CAT activity was observed in the cells cotransfected with an expression vector encoding vitamin D3 receptor (VDR) and the reporter gene. However, the synergistic CAT activity was inhibited by pretreatment with VDR antisense oligonucleotides. In addition, in a Northern blot assay, we observed 1alpha-25-(OH)2D3 synergism of TGF-beta1-induced expression of the c-jun gene in the cells transfected with the VDR expression vector and also found that the synergistic action was clearly blocked by VDR antisense oligonucleotide pretreatment. The present study strongly suggests a novel positive regulation by 1alpha-25-(OH)2D3 of TGF-beta1-induced AP-1 activity in osteoblasts via "genomic action."     

Osteotropic agents regulate the expression of osteoclast differentiation factor and osteoprotegerin in osteoblastic stromal cells

The osteoclastogenic factor of osteoblastic origin has recently been elucidated as a novel Tumor Necrosis Factor (TNF)-ligand family member, termed osteoclast differentiation factor (ODF). Using a semiquantitative RT-PCR approach, we sought to determine the mRNA expression of ODF and its decoy receptor, osteoprotegerin (OPG), in a selection of osteoblastic cell lines and in response to three factors representative of different signal transduction pathways, vitamin D receptor, protein kinase A or gp130. Each osteotropic agent, either 1,25-(OH)2D3, PTH or IL-11, promoted an increase in the ratio of ODF:OPG, with maximal stimulation occurring at 24 h, 4 h, and 8 h, respectively, and furthermore each was shown to act in a dose-dependent manner. This report establishes that osteoblastic cell lines incapable of supporting osteoclast formation have markedly reduced ODF expression and also illustrates the importance of the relative abundance of ODF compared with the levels of OPG for the induction of osteoclastogenesis.