Elevated incidence of fractures in women with invasive breast cancer.

UNLABELLED: This study evaluates the incidence of bone fractures in women with BC.We found that women with invasive breast cancer are at an increased risk for bone fractures, with fractures most commonly occurring at lower extremity and vertebral sites. The risk is further increased in women undergoing cancer therapy. INTRODUCTION: Bone loss and fractures in breast cancer have generally been attributed to aromatase inhibitor use. This study assessed the incidence of fractures after invasive breast cancer diagnosis and evaluated bone density and FRAX risk calculation at time of fracture occurrence. METHODS: Retrospective cohort study of women with invasive breast cancer [June 2003-December 2011] who participated in an academic hospital based genetic biobank. Demographic and clinical characteristics were abstracted from the electronic medical record (EMR). RESULTS: A total of 422 women with invasive breast cancer were assessed; 79 (28 %) sustained fractures during the observation period; fractures occurred at multiple skeletal sites in 27 cases (116 fractures). The incidence of fractures was 40 per 1000 person-years. Women who sustained fractures were mostly white and had a family history of osteoporosis (36.9 %, p = 0.03) or history of a prior fracture (6/79, p = 0.004). Fractures occurred 4.0 years (range 0-12 years) after cancer diagnosis. Fracture cases had femoral neck bone mineral density (BMD) of 0.72 + 0.12 g/cm(2), T-score of -1.2, that is, within the low bone mass range. Fractures most commonly occurred in lower extremities, vertebral, and wrist sites. Hip fractures accounted for 11 % of fractures, occurring at a median age of 61 years. CONCLUSIONS: Fractures occur shortly after commencing cancer therapy. Rapid bone loss associated with cancer therapy may precipitate fractures. Fractures occur at relatively higher BMD in BC. Occurrence of fractures in invasive breast cancer raises the possibility of cancer-induced impairment in bone quality.

Dose-dependent differential effects of risedronate on gene expression in osteoblasts.

Bisphosphonates have multiple effects on bone. Their actions on osteoclasts lead to inhibition of bone resorption, at least partially through apoptosis. Effects on osteoblasts vary, with modifications in the molecule and concentration both resulting in qualitatively different responses. To understand the mechanism of the differential effects of high and low bisphosphonate concentrations on osteoblast activity, we compared the effects of 10⁻8 M and 10⁻4 M risedronate on gene expression in UMR-106 rat osteoblastic cells. Two targeted arrays, an 84-gene signaling array and an 84-gene osteogeneic array were used. Gene expression was measured at 1 and 24 h. Although some genes were regulated similarly by low and high concentrations of the drug, there was also differential regulation. At 1 h, 11 genes (1 signaling and 10 osteogenesis) were solely regulated by the low concentration, and 7 genes (3 signaling, 4 osteogenesis) were solely regulated by the high concentration. At 24 h, 8 genes (3 signaling, 5 osteogenesis) were solely regulated by the low concentration and 30 genes (16 signaling and 14 osteogenesis) were solely regulated by the high concentration. Interestingly, the low, but not the high concentration of risedronate transiently and selectively upregulated several genes associated with cell differentiation. A number of genes related to apoptosis were regulated, and could be involved in effects of bisphosphonates to promote osteoblast apoptosis. Also, observed gene changes associated with decreased angiogenesis and decreased metastasis could, if they occur in other cell types, provide a basis for the effectiveness of bisphosphonates in the prevention of cancer metastases.

Antagonist minigenes identify genes regulated by parathyroid hormone through G protein-selective and G protein co-regulated mechanisms in osteoblastic cells.

Parathyroid hormone (PTH) is the major hormone regulating bone remodeling. Binding of PTH to the PTH1 receptor (PTH1R), a heterotrimeric G protein coupled receptor (GPCR), can potentially trigger multiple signal transduction pathways mediated through several different G proteins. In this study, we employed G protein antagonist minigenes inhibiting Gα(s), Gα(q) or Gα12 to selectively dissect out which of these G proteins were responsible for effects of PTH(1-34) in targeted signaling and osteogenesis arrays consisting of 159 genes. Among the 32 genes significantly regulated by 24h PTH treatment in UMR-106 osteoblastic cells, 9 genes were exclusively regulated through G(s), 6 genes were solely mediated through G(q), and 3 genes were only controlled through G12. Such findings support the concept that there is some absolute specificity in downstream responses initiated at the G protein level following binding of PTH to the PTH1R. On the other hand, 6 PTH-regulated genes were regulated by both G(s) and G(q), 3 genes were regulated by both G(s) and G12, and 3 genes were controlled by G(s), G(q) and G12. These findings indicate potential overlapping or sequential interactions among different G protein-mediated pathways. In addition, two PTH-regulated genes were not regulated through any of the G proteins examined, suggesting that additional signaling mechanisms may be involved. Selectivity was largely maintained over a 2-48-hour time period. The minigene effects were mimicked by downstream inhibitors. The dissection of the differential effects of multiple G protein pathways on gene regulation provides a more complete understanding of PTH signaling in osteoblastic cells.

G alpha12/G alpha13 subunits of heterotrimeric G proteins mediate parathyroid hormone activation of phospholipase D in UMR-106 osteoblastic cells.

PTH, a major regulator of bone remodeling and a therapeutically effective bone anabolic agent, stimulates several signaling pathways in osteoblastic cells. Our recent studies have revealed that PTH activates phospholipase D (PLD) -mediated phospholipid hydrolysis through a RhoA-dependent mechanism in osteoblastic cells, raising the question of the upstream link to the PTH receptor. In the current study, we investigated the role of heterotrimeric G proteins in mediating PTH-stimulated PLD activity in UMR-106 osteoblastic cells. Transfection with antagonist minigenes coding for small peptide antagonists to G alpha 12 and G alpha13 subunits of heterotrimeric G proteins prevented PTH-stimulated activation of PLD, whereas an antagonist minigene to G alphas failed to produce this effect. Effects of pharmacological inhibitors (protein kinase inhibitor, Clostridium botulinum exoenzyme C3) were consistent with a role of Rho small G proteins, but not of cAMP, in the effect of PTH on PLD. Expression of constitutively active G alpha12 and G alpha13 activated PLD, an effect that was inhibited by dominant-negative RhoA. The results identify G alpha12 and G alpha13 as upstream transducers of PTH effects on PLD, mediated through RhoA in osteoblastic cells.

Cyclosporine A and FK506 induce osteoclast apoptosis in mouse bone marrow cell cultures.

Studies were carried out to characterize the effects of cyclosporines and FK506 on the formation and survival of osteoclasts deriving from mouse bone marrow cultures. Cyclosporin A (CsA), cyclosporin B (CsB), cyclosporin H (CsH), and FK506 all inhibited receptor activator of NFkappaB ligand (RANKL)-stimulated tartrate-resistant acid phosphatase (TRAP) activity and generation of TRAP+ multinucleated cells in the cultures. CsA and CsG were approximately equipotent, CsH was approximately one order of magnitude less potent than the other cyclosporines, and FK506 was approximately two orders of magnitude more potent than CsA and CsG. All of the inhibitors demonstrated greater potency and efficacy on decreasing the number of TRAP+ multinucleated cells than on decreasing total TRAP activity. Further evidence that late stages were more sensitive to inhibition was obtained in experiments in which CsA was present for different segments of the RANKL-stimulated culture period. CsA was as efficacious when added for the final 2 days of a 4-day culture as when added for the entire culture period, whereas it was less effective if added for only the first 2 days of the culture. When CsA or FK506 were added for 1 day to cultures in which osteoclasts had already formed, the numbers of TRAP+ osteoclasts decreased. Treatment with CsA or FK506 produced nuclear fragmentation and disruption of the multinucleated osteoclasts and an increase in caspase-3 activity. The apoptosis inhibitor z-VAD partially prevented the inhibitory effects of CsA and FK506 on the survival of TRAP+ multinucleated cells in the cultures and also preserved the normal osteoclast morphology. The data indicate that an important component of the inhibitory effects of CsA and FK506 on marrow-derived osteoclasts is the induction of apoptosis.

Bone anabolic responses to mechanical load in vitro involve COX-2 and constitutive NOS.

Mechanical stimulation is essential for maintaining the homeostasis and architecture of connective tissues including bone. The purpose of our study was to test the importance of several potential signaling intermediates in the anabolic responses of bone to loads applied with a newly developed micromechanical loading device. Tibial bones excised from 7- to 8-day-old CD-1 mice were cyclically loaded at 1 Hz, 1000 muepsilon (microstrain) at a peak load of 100 mN. DNA and protein synthesis were evaluated by measuring the incorporation of 3H-thymidine and 14C-proline, respectively. The roles of cyclooxygenase (COX) isoforms, nitric oxide synthase (NOS) isoforms, and glutamate receptor-gated Ca2+ channeling were examined by incubating the bones in the presence of each of their specific inhibitors. The results indicate that COX-2 and constitutive NOS are important signaling molecules in the anabolic responses of neonatal tibial bone to the micromechanical load in vitro.

MicroCT quantification of in vitro bone resorption of neonatal murine calvaria exposed to IL-1 or PTH.

This study investigated how effectively a laboratory microCT (X-ray micro-computed tomography) system can quantify bone resorption in an in vitro calvarial model and how well this measure correlates with a conventional assay for calcium release (fluorometric titration). In vitro bone resorption in neonatal murine calvaria was quantified for 0.3 or 1.0 nM interleukin-1 (IL-1) or for 1.0 or 10.0 nM parathyroid hormone (PTH) treatment. Compared to control calvaria, a significantly greater fraction F of the calvarial "shell" (computed from the volumetric microCT data) was resorbed in treated calvaria of 5- to 7-day-old pups from the same litter. Excellent correlation (R2 = 0.8234) was observed between F and calcium release, and, unlike the calcium assay, the 3-D maps revealed where bone was resorbed. Mineral was preferentially lost near the sutures, and areas away from the suture were left relatively intact. MicroCT of calvaria before and after 96 h culture demonstrated that this X-irradiation neither increased control resorption nor prevented responses in the treated calvaria. Observations on calvaria from intact mice aged 1, 3, 5, 8, and 11 days showed uniformly distributed mineral (not a pronounced patchwork of "high" and "low" mineral regions) and increasing levels of mineral with age; this suggested that the spatial patterns of resorption were not related to inhomogeneities in the starting mineral distribution.

In vitro mechanical and cellular responses of neonatal mouse bones to loading using a novel micromechanical-testing device.

Mechanical stimulation is critical for the maintenance of bone architecture and bone mass. These effects are dependent on the magnitude, duration, and rate of the mechanical stimuli. The goals of the present study were to develop and optimize a micromechanical-testing device for in vitro mechanical stimulation of whole viable bones, and to identify the physical parameters of loading that elicit maximal anabolic responses. The model was the 7-8-day-old neonatal CD-1 mouse tibia. A range of cyclic strain magnitudes [500-7000 microstrain (microstrain)] and frequencies [0.2-30 hertz (Hz)] were applied to the neonatal bones. Incremental cyclic compression tests showed that the bones were nonlinearly viscoelastic. Bone stiffness and hysteresis energy dissipation were dependent on the maximum load magnitude. DNA and protein synthesis were significantly enhanced in bones that were cyclically loaded at 0.5 Hz/1000 microstrain, 0.5 Hz/2000 microstrain, or l Hz/1000 microstrain, compared to nonloaded controls. Anabolic responses were maximal at a peak load of 100 mN at l Hz/1000 microstrain. Autoradiography of the bones loaded under these conditions showed proliferation of cells at periosteal surfaces. Hysteresis energy per cycle was greatest at loads that caused the largest anabolic responses. The parameters of strain and load that elicit optimal effects on the neonatal bones are comparable to those in other systems, validating the use of the instrumentation for studying the mechanisms of the anabolic responses. The findings also suggest that hysteresis energy per cycle may be a determinant of the anabolic response of bones to mechanical stimulation.

Parathyroid hormone stimulates translocation of protein kinase C isozymes in UMR-106 osteoblastic osteosarcoma cells.

Studies with antagonists have provided evidence that protein kinase C (PKC) is involved in several of the actions of parathyroid hormone (PTH) on bone. PTH increases total PKC activity in bone and bone cells. The current studies investigated whether PTH can activate specific PKC isozymes, as demonstrated by translocation of these isozymes from cytosolic to membrane fractions. The isozymes selected for study, alpha, betaI, delta, epsilon, and zeta, were shown previously by us to be present in normal osteoblasts and several osteosarcoma-derived osteoblastic cells. UMR-106 cells, a widely used osteoblastic cell line, were selected for the current study. PKC isozymes in whole cell lysates and cell fractions were visualized by western blotting; isozyme distribution was also visualized by immunofluorescence. The total amounts of the isozymes and their relative distribution between membrane and cytosolic fractions in untreated cells were stable over a range of passages (5-20 from initial plating). In untreated cells, the concentrations of PKC alpha, betaI, and zeta were higher in the cytosol, and PKC delta and epsilon were higher in the membrane fraction. Treatment with 1 or 10 nmol/L PTH for 1 or 5 min stimulated translocation of PKC alpha and betaI, with variable effects on the other isozymes. Treatment with phorbol-12,13-dibutyrate (PDBu), 1 micromol/L for 5 min, elicited similar effects to those of PTH on PKC alpha and betaI. Treatment with PDBu for 48 h resulted in a downregulation of PKC alpha, whereas a 48 h treatment with PTH did not cause downregulation. The results indicate that PTH can affect specific PKC isozymes, providing a mechanism for differential regulation of cellular actions through this pathway.

Involvement of PKC-beta in PTH, TNF-alpha, and IL-1 beta effects on IL-6 promoter in osteoblastic cells and on PTH-stimulated bone resorption.

Protein kinase C (PKC) has been shown to be activated by parathyroid hormone (PTH) in osteoblasts. Prior evidence suggests that this activation mediates responses leading to bone resorption, including production of the osteoclastogenic cytokine interleukin-6 (IL-6). However, the importance of specific PKC isozymes in this process has not been investigated. A selective antagonist of PKC-beta, LY379196, was used to determine the role of the PKC-beta isozyme in the expression of IL-6 in UMR-106 rat osteoblastic cells and in bone resorption in fetal rat limb bone organ cultures. PTH, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta) induced translocation of PKC-alpha and -beta(I) to the plasma membrane in UMR-106 cells within 5 min. The stimulation of PKC-beta(I) translocation by PTH, TNF-alpha or IL-1 beta was inhibited by LY379196. In contrast, LY379196 did not affect PTH, TNF-alpha-, or IL-1 beta-stimulated translocation of PKC-alpha. PTH, TNF-alpha, and IL-1 beta increased luciferase expression in UMR-106 cells transiently transfected with a -224/+11 bp IL-6 promoter-driven reporter construct. The IL-6 responses were also attenuated by treatment with LY379196. Furthermore, LY379196 inhibited bone resorption elicited by PTH in fetal rat bone organ cultures. These results indicate that PKC-beta(I) is a component of the signaling pathway that mediates PTH-, TNF-alpha-, and IL-1 beta-stimulated IL-6 expression and PTH-stimulated bone resorption.

Stimulation of interleukin-6 promoter by parathyroid hormone, tumor necrosis factor alpha, and interleukin-1beta in UMR-106 osteoblastic cells is inhibited by protein kinase C antagonists.

To investigate the level at which protein kinase C (PKC) regulates expression of interleukin-6 (IL-6) in osteoblastic cells, effects of several PKC antagonists and PKC down-regulation by phorbol ester were studied in UMR-106 osteoblastic cells that had been transiently transfected with a -224/+11-base pair (bp) IL-6 promoter coupled to a luciferase reporter. Parathyroid hormone (PTH) elicited a dose-dependent stimulation of the IL-6 promoter expression, with significant increases produced by 5 h of treatment with concentrations of PTH as low as 10(-14) M. The increase in IL-6 promoter expression was inhibited by the PKC antagonists GF109203X, 30 nM to 1 microM, and calphostin C, 250 nM. Prior down-regulation of PKC with 100 nM phorbol-12,13-dibutyrate (PDBU) for 48 h inhibited the PTH effect as well as the smaller stimulatory effects elicited by tumor necrosis factor alpha (TNF-alpha), 10(-9)-10(-8) M, and by IL-1beta, 1-10 ng/ml. In contrast to these findings, the stimulatory effects of PTH, TNF-alpha, and IL-1beta on the IL-6 promoter expression were enhanced by staurosporine. Treatment with GF109203X or down-regulation of PKC with PDBU prevented the stimulatory effects of staurosporine. PKC activity was increased by staurosporine. The findings with staurosporine are consistent with our earlier observations that this agent enhances the calcium signaling and bone resorption elicited by PTH. The studies support the role of PKC in the stimulatory effects of PTH, TNF-alpha, and IL-1beta on IL-6 expression.

Mutual up-regulation of thyroid hormone and parathyroid hormone receptors in rat osteoblastic osteosarcoma 17/2.8 cells.

PTH and thyroid hormone (T(3)) stimulate anabolic and catabolic processes in bone predominantly by acting on osteoblasts. Both inadequate and excessive secretion of either hormone can result in clinical bone disorders. In addition, T(3) and PTH related peptide (PTHrP) have multiple effects on a wide number of other tissues modulating both cell differentiation and proliferation. To address the question of whether there might be functional mutual regulation of T(3) receptors (TR) and PTH/PTHrP receptors (PTHR), we studied their expression and receptor-mediated intracellular effects in rat osteoblastic osteosarcoma (ROS) 17/2.8 cells. PTHR were up-regulated by T(3) pretreatment (10(-)(10)-10(-)(6) M) in ROS 17/2.8 cells in a dose-dependent manner. T(3) pretreatment increased both PTH-induced cyclic AMP response element binding protein (CREB) phosphorylation and PTH-induced intracellular calcium transients, and further decreased PTH-induced down-regulation of alkaline phosphatase activity, suggesting that there are functional consequences of the PTHR up- regulation. Pretreatment with PTH (10(-)(10)-10(-)(6) M) or PTHrP (10(-)(9) M) for 3-4 days resulted in a dose-dependent up-regulation of TR in ROS 17/2.8 cells. cAMP analogues or a calcium ionophore were able to mimic the effect of PTH on TR binding, suggesting that either the cAMP-signaling pathway or Ca(2+) could be involved in PTH-induced up-regulation of the TR. These observations provide a novel example of mutual interactions between nuclear receptors and membrane receptors and may have significant implications for the regulation of bone remodeling in health and disease.

Phosphatidylcholine-specific phospholipase C inhibitor, tricyclodecan-9-yl xanthogenate (D609), increases phospholipase D-mediated phosphatidylcholine hydrolysis in UMR-106 osteoblastic osteosarcoma cells.

Our previous studies have shown that parathyroid hormone (PTH) stimulates phosphatidylcholine (PC) hydrolysis by phospholipase D (PLD) and transphosphatidylation in UMR-106 osteoblastic cells. To determine whether phospholipase C (PLC) is also involved in the PTH-mediated PC hydrolysis, we used the inhibitor, tricyclodecan-9-yl xanthogenate (D609), a putatively selective antagonist of this pathway. Consistent with this proposed mechanism, D609 decreased (3)H-phosphocholine in extracts from UMR-106 cells prelabeled with (3)H-choline. Unexpectedly, D609 enhanced PC hydrolysis and transphosphatidylation, suggesting that either there was a compensatory increase in PLD activity when PLC was inhibited, or that D609 directly increased PLD activity. The D609-stimulated increase in PC hydrolysis was rapid, being seen as early as 2 min. The effect of D609 was temperature-sensitive, consistent with an enzymatic mechanism. The D609-stimulated increase in PC hydrolysis was PKC-independent, based upon the lack of effect of down-regulation of PKC by phorbol 12,13-dibutyrate on the response. The studies reveal a novel action of this inhibitor on signaling in osteoblastic cells which might influence downstream responses.

Divergent effects of protein kinase C (PKC) inhibitors staurosporine and bisindolylmaleimide I (GF109203X) on bone resorption.

Activation of protein kinase C (PKC) has been suggested to play a role in bone resorption. However, phorbol esters, which activate PKC, have been reported to have both stimulatory and inhibitory effects on bone resorption. To study the role of PKC in bone resorption further, we have measured calcium release elicited by bone-resorbing hormones from isolated bones incubated with the PKC inhibitors staurosporine (ST) and the more PKC-selective ST analog bisindolylmaleimide I (GF109203X; GF). In fetal rat limb bone organ cultures, ST (1 microM) or GF (1 microM) significantly reduced the bone resorption induced by maximal concentrations of parathyroid hormone (PTH). However, when submaximal concentrations of PTH were used, lower concentrations of the two antagonists had divergent effects. GF (20-300 nM) acted solely as an antagonist, whereas ST (10-100 nM) significantly enhanced resorptive responses to PTH. ST also enhanced the bone resorption elicited by alpha-thrombin, tumor necrosis factor-alpha (TNF-alpha), and thyroxin (T4). ST alone had small stimulatory effects in some experiments. GF prevented the stimulatory effects of ST alone as well as the enhancing effect of ST on PTH-stimulated resorption. The divergent effects of ST and GF on the responses of bone to low concentrations of PTH and the ability of GF to antagonize the stimulatory effects of ST suggest that PKC isozymes have complex and even antagonistic effects on bone resorption.

Endothelin-stimulated Ca(2+)signaling and endothelin receptor expression are decreased by parathyroid hormone treatment in UMR-106 osteoblastic osteosarcoma cells.

Modulation of endothelin (ET-1)-induced [Ca(2+)](i)transients and receptor expression by parathyroid hormone (PTH) was studied in UMR-106 osteoblastic osteosarcoma cells. Ca(2+)signaling was assessed with Fura-2, and ET receptor mRNA expression was determined using ET(A)- and ET(B)-specific primers and RT-PCR amplification. ET-1 binding in UMR-106 cell membranes was also measured. PTH pretreatment for 8 h decreased the [Ca(2+)](i)transients elicited by ET-1 and by the ET(B)-selective agonist sarafotoxin 6c (S6c). When ET(B)receptors were desensitized by pretreatment with S6c or blocked with the ET(B)-selective antagonist BQ-788, the remaining ET(A)component of the signal was also decreased by PTH pretreatment. In contrast, [Ca(2+)](i)transients elicited by PGF(2alpha)and ionomycin were increased following PTH pretreatment, indicating that the effect of PTH to decrease ET-1-stimulated transients was selective. PTH pretreatment also decreased [(125)I]ET-1 binding and ET(A)and ET(B)mRNA, with maximal effects at approximately 8 h. ET-1 was not detectable in medium from either control or PTH treated UMR-106 cultures, suggesting that the decreased expression of ET receptors was not due to enhanced ET production and subsequent homologous desensitization. The downregulation of ET receptors in osteoblasts by PTH pretreatment may serve as a homeostatic mechanism in bone.

Serum insulin-like growth factor-I, insulin-like growth factor binding proteins, and bone mineral content in hyperthyroidism.

The mechanism by which thyroid hormones promote bone growth has not yet been elucidated. In vitro, thyroid hormones stimulate insulin-like growth factor-I (IGF-I) production by osteoblasts, which is important for the anabolic effects of the hormone on bone. To determine whether the IGF-I/IGF binding protein (IGFBP) profile is affected when thyroid hormone production is altered in vivo, we studied 36 women who had recently been diagnosed with hyperthyroidism (age: 29-67 years; 19 with Graves' disease, 17 with toxic nodular goiter) and 36 age-matched healthy women as controls. Serum IGF-I, and its binding proteins (IGFBP-3, IGFBP-4, and IGFBP-5), as well as bone mineral density (BMD) at the lumbar spine, femoral neck, and radius midshaft were measured before and 1 year after antithyroid (methimazole) treatment. Serum IGF-I levels were significantly increased in the hyperthyroid patients before treatment (214 +/- 18.2 ng/mL vs. 145 +/- 21.3 ng/mL; p < 0.05). There was no difference in IGF-I levels of patients with Graves' disease and toxic nodular goiter. Serum IGF-I concentrations returned to normal after treatment with methimazole. Serum IGFBP-3 and IGFBP-4 values were significantly elevated in the hyperthyroid group before treatment (3960 +/- 220 ng/mL and 749.7 +/- 53.1 ng/mL vs. 2701 +/- 180 ng/mL and 489.9 +/- 32.4 ng/mL; p < 0.05 and p < 0.01, respectively) and were reduced to those of controls after treatment. Serum IGFBP-5 of hyperthyroid subjects was not different from that of controls either before or after therapy. Serum free thyroxine showed a positive correlation with serum levels of IGF-I (r = 0.73, p < 0.05), IGFBP-3 (r = 0.59, p < 0.05), and IGFBP-4 (r = 0.67, p < 0.05) but not IGFBP-5. BMD at the radius midshaft was significantly lower in hyperthyroid patients at the start of the study and showed a positive correlation with serum IGF-I (r = 0.58; p < 0.001) and a negative correlation with IGFBP-4 (r = -0.61; p < 0.05). Radius BMD showed a 7.2% increase in the hyperthyroid group after 1 year of methimazole treatment, and the correlation between BMD and serum IGF-I disappeared. Our data indicate that thyroid hormones may influence the IGF-I/IGFBP system in vivo in hyperthyroidism. The anabolic effects of increased levels of IGF-I may be limited in hyperthyroidism due to the increases of inhibitory IGFBPs that can counteract the anabolic effects and contribute to the observed net bone loss.

Protein kinase C involvement in interleukin-6 production by parathyroid hormone and tumor necrosis factor-alpha in UMR-106 osteoblastic cells.

The cytokine interleukin-6 (IL-6) is increased in bone and bone cells by several resorptive stimuli, including parathyroid hormone (PTH), IL-1beta, and tumor necrosis factor-alpha (TNF-alpha). The current studies were designed to determine the contribution of the protein kinase C (PKC) signaling pathway to the effects of these three agents to increase IL-6 in UMR-106 rat osteoblastic cells. Cells were pretreated with vehicle (dimethylsulfoxide [DMSO]) or the phorbol ester, phorbol 12,13-dibutyrate (PDB; 300 nM) for 48 h to down-regulate phorbol-sensitive PKC isozymes. Either PTH (0.1-10 nM), IL-1beta (0.1-10 nM), or TNF-alpha (5 nM and 10 nM) was then added for 24 h in the continued presence of vehicle or PDB. PKC isozymes were visualized by Western immunoblotting and IL-6 was determined by bioassay. PDB pretreatment caused a partial down-regulation of the conventional alpha-PKC and betaI-PKC isozymes and complete down-regulation of the novel delta-isoenzyme and epsilon-isozymes but it had no effect on the atypical zeta-PKC isozyme. PDB pretreatment reduced IL-6 responses to 5 nM and 10 nM PTH by 61% and 33%, respectively, reduced IL-6 responses to 5nM and 10 nM TNF-a by 54% and 42%, respectively, and failed to inhibit the IL-6 responses to 0.1-10 nM IL-1beta. The PDB pretreatment protocol significantly enhanced PTH-stimulated cyclic adenosine monophosphate (cAMP) production. The PKC inhibitor calphostin C also decreased IL-6 responses to PTH. Thus, in this osteoblast cell line, the PKC pathway is an important component of the signaling pathway for the IL-6 production stimulated by PTH and TNF-alpha but not that from IL-1beta.

Compactin suppresses bone resorption by inhibiting the fusion of prefusion osteoclasts and disrupting the actin ring in osteoclasts.

Compactin (mevastatin), which inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, and thus biosynthesis of cholesterol and the prenylation of proteins, inhibits osteoclastic bone resorption. Although it has been suggested that compactin inhibits bone resorption by inducing apoptosis of osteoclasts, the pathway by which compactin inhibits resorption has not been established. We investigated the effect of compactin on the differentiation of osteoclasts and the relationship between the morphological changes elicited by compactin and its inhibitory effect on bone resorption. Compactin inhibited the differentiation of osteoclasts, interfering with the fusion process by which prefusion osteoclasts (pOCs) develop into multinucleated osteoclast-like cells (OCLs), and also disrupted the actin ring of OCLs. The potency of compactin to inhibit fusion of pOCs and to disrupt the actin ring of OCLs corresponded to that of compactin to inhibit bone resorption. The effects of compactin were prevented by the addition of MVA lactone or its downstream products farnesylpyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP) but not by squalene. Apoptosis of OCLs was not induced by the concentration of compactin that inhibited fusion of pOCs and disrupted the actin ring. The normal process of pOC fusion and the integrity of the actin ring were restored by the withdrawal of compactin from the cultures after they had been treated with compactin for 24 h, but they were not restored by the addition of zVAD-fmk, a caspase inhibitor. Compactin also reversibly inhibited interleukin-1beta (IL-1beta)-, 1alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3)-, and parathyroid hormone (PTH)-stimulated 45Ca release in bone organ cultures. Our results indicate that the inhibitory effects of compactin on bone resorption result from the inhibition of fusion of pOCs into OCLs and disruption of actin ring in OCLs and that apoptosis of OCLs is not necessary for these inhibitory effects of compactin. These effects of compactin are likely to be a consequence of the inhibition of prenylation of proteins that play an important role in the fusion of pOCs and in maintaining actin ring integrity in OCLs.

Insulin-like growth factor I production is essential for anabolic effects of thyroid hormone in osteoblasts.

Thyroid hormone (T3) and insulin-like growth factor I (IGF-I) are critical regulators of skeletal function. T3 increases IGF-I production in bone. To assess the potential role of IGF-I as a mediator of T3 actions, we characterized phenotypic markers of osteoblast activity in two osteoblast models, normal mouse osteoblasts and MC3T3-E1 cells, exposed to T3 alone or under conditions that interfere with IGF-I actions. T3 significantly increased osteoblast 3H-proline incorporation, alkaline phosphatase (ALP), and osteocalcin. Both alphaIR3, a neutralizing monoclonal antibody to the IGF-I receptor, and JB1, an IGF-I analogue antagonist, attenuated the stimulatory effects of T3. T3 effects also were decreased in cells transfected with antisense oligonucleotide (AS-ODN) to the IGF-I receptor gene. Both IGF-I and T3 had mitogenic effects that were inhibited by the antagonists. IGF-I by itself did not stimulate 3H-proline incorporation, ALP, and osteocalcin in the models used, revealing that although IGF-I is essential for the anabolic effects of T3, it acts in concert with other factors to elicit these phenotypic responses.

Staurosporine enhances parathyroid hormone-induced calcium signal in UMR-106 osteoblastic cells.

Parathyroid hormone (PTH) treatment of bone and kidney-derived cells not only activates adenylyl cyclase but also increases intracellular free calcium, and translocates protein kinase C (PKC) from cytosol to plasma membranes. We have found that acute phorbol ester pretreatment significantly decreases PTH-induced calcium transients and the effect of phorbol ester was antagonized by staurosporine (ST). Although the major effect of ST in that study was the reversal of the action of phorbol ester, it appeared that ST may also have promoted the effect of PTH directly. To further investigate the observation, we examined the effect of ST on the intracellular calcium transients induced by PTH and alpha-thrombin (alpha-TH). For calcium transient experiments, UMR-106 cells were loaded with 2 mM fluo-acetoxymethylester for 30 min at room temperature. The cells were then washed and suspended in buffer containing 1 mM calcium. Fluorescence was detected at 530 nm, with excitation at 505 nm. ST alone did not cause calcium transients, but enhanced the transients elicited by PTH when added 5 min before the hormone. Another protein kinase inhibitor H-7 likewise enhanced the calcium responses elicited by PTH, while genistein did not affect PTH response. Calcium transients elicited by alpha-TH were also enhanced by ST. The results suggest that there might be tonically activated endogenous protein kinase(s) which inhibit calcium signaling of some calcemic agents.