Inhibit Osteoblast Activity Research Articles

Expression of molecular markers for bone formation increases during experimental acute otitis media

Bony tissues are integral parts of the function of the middle ear and the protection of adjacent vital structures. To explore the reaction of middle ear bone to acute otitis media, rats were challenged with Streptococcus pneumoniae and Haemophilus influenzae. Local changes were monitored for up to 1 month. After reverse transcription, competitive polymerase chain reaction was used to determine the expression levels of two molecular markers of bone formation, osteocalcin and procollagen I, and the two cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α, in the bone. Middle ear bone responded rapidly to bacterial challenge, and the reaction depended upon the causative agent. On day 1, IL-6 and TNF-α transcripts were detected in the bone from all middle ears. After a short period of decreased expression of osteocalcin, during which the otitis diagnosis could not be made clinically, the levels of bone formation markers increased dramatically. The maximum levels of these markers were reached on days 6 and 14 for animals challenged with H. influenzae and pneumococci, respectively. Infections induced by pneumococci had a longer duration, and after the initial phase the production of osteocalcin and procollagen transcript were significantly higher in the pneumococcus-infected animals. The results indicate that even in an uncomplicated infection, the bone of the bulla reacts to an acute otitis media with a short period of inhibited osteoblast activity followed by a longer period of new bone formation.

Effect of the vitamin D analogues paricalcitol and calcitriol on bone mineral in vitro

Paricalcitol (19-nor-1,25-dihydroxyvitamin D2 ), a new vitamin D analogue, recently became available for the treatment of hyperparathyroidism in patients with end-stage renal disease. It is safe and effective in suppressing parathyroid hormone, with apparently less propensity for hypercalcemia than calcitriol (1,25-dihydroxyvitamin D3 ). However, the mechanism of action on bone has not been fully elucidated. This study compares the effects of paricalcitol and calcitriol on the bone mineral. Neonatal (5- to 7-day-old) mouse calvariae were incubated in the absence or presence of either paricalcitol or calcitriol for 48 hours, and calcium flux, osteocalcin and acid and alkaline phosphatase activity, and interleukin-6 (IL-6) release were determined. Increasing concentrations of both calcitriol and paricalcitol increased calcium efflux. At lower concentrations, paricalcitol had no effect on acid phosphatase activity; however, at 10-8 mol/L, paricalcitol caused a significant increase similar to that of calcitriol at 10-9 mol/L. Increasing concentrations of paricalcitol had no effect on alkaline phosphatase activity, whereas calcitriol (10-8 mol/L) caused significant inhibition. At low concentrations, paricalcitol had no effect on osteocalcin release; however, at 10-8 mol/L, both compounds significantly increased osteocalcin production. Neither compound had an effect on IL-6 release. These data show that: (1) at low concentrations, both compounds induce a similar calcium efflux from cultured bone; (2) at low concentrations, paricalcitol has no effect on osteocalcin or acid and alkaline phosphatase activity; (3) at greater concentrations, paricalcitol and calcitriol have similar effects on acid phosphatase and osteocalcin activity; (4) calcitriol, but not paricalcitol, inhibits alkaline phosphatase release; and (5) the bone-resorbing effect of both compounds is independent of IL-6 release. Thus, although both compounds have similar effects on calcium efflux from bone, at therapeutic concentrations, paricalcitol does not seem to inhibit osteoblast activity. This may explain, in part, the lower calcemic effect of paricalcitol.

Hepatocyte Growth Factor (HGF) Induces Interleukin-11 Secretion From Osteoblasts: A Possible Role for HGF in Myeloma-Associated Osteolytic Bone Disease

AbstractMultiple myeloma is associated with unbalanced bone remodeling causing lytic bone lesions. Interleukin-11 (IL-11) promotes osteoclast formation and inhibits osteoblast activity and may, thus, be one factor involved in cancer-induced bone destruction. We have previously shown that myeloma cells produce hepatocyte growth factor (HGF). We now report that HGF induces IL-11 secretion from human osteoblast-like cells and from the osteosarcoma cell lines Saos-2 and HOS. In coculture experiments, both the myeloma cell line JJN-3 and primary myeloma cells from 3 patients induced IL-11 secretion from osteoblasts, whereas no induction was observed with the non-HGF producing myeloma cell line OH-2. Enhanced IL-11 induction was observed with physical contact between osteoblasts and myeloma cells as compared with experiments in which contact was prohibited by tissue inserts. Anti-HGF serum strongly reduced the myeloma cell-induced IL-11 secretion. Furthermore, we show that JJN-3 cells express HGF on the cell-surface. Removal of surface-bound HGF on JJN-3 cells reduced IL-11 production induced in cocultures. Transforming growth factor β1 and IL-1 potentiated the effect of HGF on IL-11 secretion, whereas an additive effect was observed with tumor necrosis factor. Thus, myeloma-derived HGF can influence the bone marrow environment both as a soluble and a surface-bound factor. Furthermore, HGF emerges as a possible factor involved in myeloma bone disease by its ability to induce IL-11.

Hepatocyte Growth Factor (HGF) Induces Interleukin-11 Secretion From Osteoblasts: A Possible Role for HGF in Myeloma-Associated Osteolytic Bone Disease

Multiple myeloma is associated with unbalanced bone remodeling causing lytic bone lesions. Interleukin-11 (IL-11) promotes osteoclast formation and inhibits osteoblast activity and may, thus, be one factor involved in cancer-induced bone destruction. We have previously shown that myeloma cells produce hepatocyte growth factor (HGF). We now report that HGF induces IL-11 secretion from human osteoblast-like cells and from the osteosarcoma cell lines Saos-2 and HOS. In coculture experiments, both the myeloma cell line JJN-3 and primary myeloma cells from 3 patients induced IL-11 secretion from osteoblasts, whereas no induction was observed with the non-HGF producing myeloma cell line OH-2. Enhanced IL-11 induction was observed with physical contact between osteoblasts and myeloma cells as compared with experiments in which contact was prohibited by tissue inserts. Anti-HGF serum strongly reduced the myeloma cell-induced IL-11 secretion. Furthermore, we show that JJN-3 cells express HGF on the cell-surface. Removal of surface-bound HGF on JJN-3 cells reduced IL-11 production induced in cocultures. Transforming growth factor β1 and IL-1 potentiated the effect of HGF on IL-11 secretion, whereas an additive effect was observed with tumor necrosis factor. Thus, myeloma-derived HGF can influence the bone marrow environment both as a soluble and a surface-bound factor. Furthermore, HGF emerges as a possible factor involved in myeloma bone disease by its ability to induce IL-11.

Evidence that conditionally immortalized human osteoblasts express an osteocalcin receptor

Osteocalcin (OC) is an abundant noncollagenous bone matrix protein, yet its function is largely unknown. However, targeted ablation of two OC genes in mice lead to increased bone formation (Ducy et al. Nature 382:448–452; 1996). This implied that OC inhibits osteoblast activity, and that these cells express an OC receptor. In order to characterize the putative OC receptor, we used the Cytosensor microphysiometer to measure responses of a proliferative-stage, conditionally immortalized human osteoblast cell line (HOB-03-C5) to purified bovine OC (bOC). The Cytosensor measures a change in the extracellular acidification rate, which is primarily a measurement of metabolic activity. Treatment of the HOB cells for 5–60 sec with 0.17 μmol/L bOC generated a time-dependent, transient increase in the acidification rate that became optimal after 25 sec. Likewise, treatment of the cells for 25 sec with 0.021 to 1.9 μmol/L bOC caused a dose-dependent 70% increase in the acidification rate. Pretreatment of the cells for 2 h with inhibitors of adenylyl cyclase, phospholipase C, and intracellular calcium release inhibited the response of the cells to bOC by 50%–100%, which suggested that the putative OC receptor was coupled to a G-protein. These observations from the Cytosensor were confirmed by measuring intracellular cyclic-adenosine monophosphate (cAMP) concentrations in response to bOC. Treatment of the cells for 10 min with bOC decreased basal cAMP levels by 65% in a dose-dependent manner with an IC 50 of 0.22 μM. However, cotreatment of the cells with forskolin, which activates adenylyl cyclase, blunted this suppression. Moreover, pretreatment of the cells with pertussis toxin for 48 h, which inhibits Gαi proteins, reversed the suppressive effects of bOC on cAMP production. Treatment of the HOB cells for 48 h with 0.19 to 1.5 μmol/L bOC caused a dose-dependent 40% decrease in alkaline phosphatase activity with an IC 50 of 0.21 μmol/L, which suggested that OC may inhibit HOB activity. Finally, although the maturation stage, conditionally immortalized HOB-02-C1 cells also responded to bOC as measured by the Cytosensor, two osteosarcoma cell lines, SaOS-2 and ROS 17/2.8, exhibited a 5- to 10-fold lower response to the bone matrix protein, suggesting that the putative OC receptor was downregulated in these cells. However, all of these bone cell lines responded to parathyroid hormone treatment. In conclusion, these results provide evidence that the HOB cells express an OC receptor, and that this receptor appears to be coupled to a Gαi-protein.

The effects of acid glycosaminoglycans on neonatal calvarian cultures — A role of keratan sulfate in morquio syndrome?

Morquio syndrome (mucopolysaccharidosis IV) presents with multiple bone dysplasia and is characterized by the inability to degrade keratan sulfate due to deficient N-acetylgalactosamine-6-sulfate sulfatase in Morquio A syndrome and deficient beta-D-galactosidase in Morquio B syndrome. The aim of our study was to investigate into the pathogenetic mechanism as it is not clear whether the accumulation of keratan sulfate is toxic for osteoblasts or inhibits osteoblast activity as e.g. bone resorption. The glycosaminoglycans keratan sulfate, heparan sulfate, dermatan sulfate, chondroitin-4,6-sulfate and hyaluronic acid were tested in rat neonatal calvarian cultures for their effects on bone resorption, osteoblast activity and toxicity. Bone resorption was evaluated by calcium release into the medium, osteoblast activity by the determination of alkaline phosphatase and toxicity by measuring lactate dehydrogenase in the culture media. Keratan sulfate had no effect on bone resorption but inhibited osteoblast activity at the low, nontoxic concentration of 10 ng per ml organ culture supernatant significantly (p < 0.05). At a concentration of 100 ng per ml keratan sulfate revealed toxic effects as reflected by significantly (p < 0.05) elevated lactate dehydrogenase activity. None of the other glycosaminoglycans inhibited osteoblast activities. Heparan sulfate showed at toxic levels (10 μg per ml supernatant) significantly increased bone resorption (p < 0.05) accompanied by increased alkaline phosphatase activity. The specific keratan sulfate effects of inhibiting osteoblast activity and toxicity towards bone, which were never tested before, suggest a role for this glycosaminoglycan in the pathogenesis of bone dysplasia in Morquio syndrome.

The Effect of Low‐Dose Methotrexate on Bone Metabolism and Histomorphometry in Rats

To ascertain the effects of low-dose methotrexate (MTX) on bone metabolism and histomorphometry in rats. Female Sprague-Dawley rats (6 months old, n = 42) were divided into the following 4 groups: intraperitoneal (IP) injections of MTX, with and without ovariectomy, and IP saline (controls), with and without ovariectomy. Injections were given for 16 weeks. The MTX dose was equivalent to a standard dose for rheumatoid arthritis in humans that would yield similar serum MTX levels (0.6 +/- 0.1 mumoles). Bone formation (assessed by serum alkaline phosphatase and osteocalcin levels and histomorphometry) was significantly lower in the MTX groups, and bone resorption (assessed by urinary hydroxyproline levels and histomorphometry) was increased in the MTX groups. Bone mass was significantly diminished in the MTX groups. Prolonged administration of low-dose MTX in rats causes significant osteopenia via suppression of osteoblast activity and stimulation of osteoclast recruitment, which results in increased bone resorption.

Coupling of resorption and formation on bone remodeling sequence in orthodontic tooth movement: A histochemical study

Rat's molars were submitted to orthodontic tooth movement. Bone formation areas were detected using lead-labeling technique. Osteoclasts and osteoblasts were detected by enzyme histochemistry using Tartrate resistant Acid phosphatase (TRACPase) and Alkaline phosphatase (ALPase) to determine simultaneously and mark the 2 types of cells on a same section. The sites selected for study were pressure/distal, tension/mesial and transitional areas of second molars. The results showed that: orthodontic force activated bone remodeling sequence throughout the alveolar bone; slight new bone formation was observed on the cement line on the pressure side. ALPase-positive cells were detected on the pressure side neighboring osteoclasts. On the tension side, bone formation was enhanced in the protrusions whereas both resorption and formation were observed in the depressions. In the transitional area, cellular sequence from osteoclastic bone resorption to bone formation was revealed over the cement line. These findings demonstrated that: coupling phenomena occur on the pressure side but with inhibited osteoblast activity. Bone formation on the tension side involves both promotion of bone formation by the traction force; bone remodeling sequence is established on the tension side by the interaction between osteoclastic bone resorption and bone formation that takes place in the depressions; Coupling phenomena occur in the transitional area as well. Our findings on the pressure side led to the consideration that osteoblastic cells in periodontal ligament would be involved in the regulation of osteoclastic bone resorption.