Altered chondrocyte differentiation, matrix mineralization and MEK-Erk1/2 signaling in an INPPL1 catalytic knock-out mouse model of opsismodysplasia.

Opsismodysplasia (OPS) is a rare but severe autosomal recessive skeletal chondrodysplasia caused by inactivating mutations in the Inppl1/Ship2 gene. The molecular mechanism leading from Ship2 gene inactivation to OPS is currently unknown. Here, we used our Ship2Δ/Δ mouse expressing reduced amount of a catalytically-inactive SHIP2 protein and a previously reported SHIP2 inhibitor to investigate growth plate development and mineralization in vivo, ex vivo and in vitro. First, as observed in OPS patients, catalytic inactivation of SHIP2 in mouse leads to reduced body length, shortening of long bones, craniofacial dysmorphism, reduced height of the hyperthrophic chondrocyte zone and to defects in growth plate mineralization. Second, intrinsic Ship2Δ/Δ bone defects were sufficient to induce the characteristic OPS alterations in bone growth, histology and mineralization ex vivo. Third, expression of osteocalcin was significantly increased in SHIP2-inactivated chondrocyte cultures whereas production of mineralized nodules was markedly decreased. Targeting osteocalcin mRNA with a specific shRNA increased the production of mineralized nodules. Fourth, levels of p-MEK and p-Erk1/2 were significantly increased in SHIP2-inactivated chondrocytes in response to serum and IGF-1, but not to FGF2, as compared to control chondrocytes. Treatment of chondrocytes and bones in culture with a MEK inhibitor partially rescued the production of mineralized nodules, the size of the hypertrophic chondrocyte zone and bone growth, raising the possibility of a treatment that could partially reduce the phenotype of this severe condition. Altogether, our results indicate that Ship2Δ/Δ mice represent a relevant model for human OPS. They also highlight the important role of SHIP2 in chondrocytes during endochondral ossification and its different differentiation steps. Finally, we identified a role of osteocalcin in mineralized nodules production and for the MEK-Erk1/2 signaling pathway in the OPS phenotype.

Effects of warfarin on biological processes other than haemostasis: A review.

Warfarin is the world's most widely used anticoagulant drug. Its anticoagulant activity is based on the inhibition of the vitamin K-dependent (VKD) step in the complete synthesis of a number of blood coagulation factors that are required for normal blood coagulation. Warfarin also affects synthesis of VKD proteins not related to haemostasis including those involved in bone growth and vascular calcification. Antithrombotic activity of warfarin is considered responsible for some aspects of its anti-tumour activity of warfarin. Some aspects of activities against tumours seem not to be related to haemostasis and included effects of warfarin on non-haemostatic VKD proteins as well as those not related to VKD proteins. Inflammatory/immunomodulatory effects of warfarin indicate much broader potential of action of this drug both in physiological and pathological processes. This review provides an overview of the published data dealing with the effects of warfarin on biological processes other than haemostasis.

High density lipoprotein modulates osteocalcin expression in circulating monocytes: a potential protective mechanism for cardiovascular disease in type 1 diabetes.

BACKGROUND: Cardiovascular disease (CVD) is a major cause of mortality in type 1 diabetes (T1D). A pro-calcific drift of circulating monocytes has been linked to vascular calcification and is marked by the surface expression of osteocalcin (OCN). We studied OCN+ monocytes in a unique population with ≥50 years of T1D, the 50-Year Joslin Medalists (J50M). METHODS: CD45 bright/CD14+/OCN+ cells in the circulating mononuclear blood cell fraction were quantified by flow cytometry and reported as percentage of CD45 bright cells. Mechanisms were studied by inducing OCN expression in human monocytes in vitro. RESULTS: Subjects without history of CVD (n = 16) showed lower levels of OCN+ monocytes than subjects with CVD (n = 14) (13.1 ± 8.4% vs 19.9 ± 6.4%, p = 0.02). OCN+ monocytes level was inversely related to total high density lipoprotein (HDL) cholesterol levels (r = -0.424, p = 0.02), large (r = -0.413, p = 0.02) and intermediate (r = -0.445, p = 0.01) HDL sub-fractions, but not to small HDL. In vitro, incubation with OxLDL significantly increased the number of OCN+ monocytes (p < 0.01). This action of OxLDL was significantly reduced by the addition of HDL in a concentration dependent manner (p < 0.001). Inhibition of the scavenger receptor B1 reduced the effects of both OxLDL and HDL (p < 0.05). CONCLUSIONS: Low OCN+ monocytes levels are associated with lack of CVD in people with long duration T1D. A possible mechanism for the increased OCN+ monocytes could be the elevated levels of oxidized lipids due to diabetes which may be inhibited by HDL. These findings suggest that circulating OCN+ monocytes could be a marker for vascular disease in diabetic patients and possibly modified by HDL elevation.

Berberine promotes bone marrow-derived mesenchymal stem cells osteogenic differentiation via canonical Wnt/ß-catenin signaling pathway.

Berberine (BBR) has recently been reported to be extensively used for musculoskeletal disorders such as osteoporosis through enhancing osteogenic differentiation, inhibiting osteoclastogenesis and bone resorption and repressing adipogenesis. Although canonical Wnt signaling plays a crucial role in suppressing bone marrow-derived mesenchymal stem cells (MSCs) commitment to the chondrogenic and adipogenic lineage and enhancing osteogenic differentiation, no previous reports have shown an association between BBR-induced osteogenesis and Wnt/ß-catenin signaling pathway. In this study, we aimed to investigate the stimulatory effect and the mechanism of BBR on osteogenic differentiation of human bone marrow-derived MSCs. MSCs were isolated from bone marrow specimens and treated with different concentration of BBR. Cell viability was measured by the WST-8 assay. Effects of BBR on osteogenic differentiation of MSCs were assessed by von Kossa staining, ALP staining and ALP activity. Osteogenic specific genes, chondrogenic and adipogenic related marker genes were determined by quantitative real-time polymerase chain reaction analysis. Western blot and Immunofluorescence staining were performed to analyze OCN and OPN, and ß-catenin expression in the presence or absence of BBR combined with DKK-1 or ß-catenin siRNA transfection. Increasing concentration of BBR (3, 10 and 30 µM) promoted osteogenic differentiation and osteogenic genes expression after incubation for various days compared with DMSO group, whereas expression levels of chondrogenic and adipogenic related marker genes were dramatically suppressed. After treated with 10µM BBR for 7 days, ß-catenin, OPN and OCN expression were significantly induced, which could be effectively suppressed by the addition of DKK-1 or ß-catenin siRNA ß-catenin. Interestingly, the expression level of Runx2 gene was also decreased by inhibiting the transduction of Wnt/ß-catenin signaling. These findings suggest that BBR can stimulate osteogenic differentiation of MSCs not only by enhancing Runx2 expression but also by activating canonical Wnt/ß-catenin signaling pathway, and canonical Wnt/ß-catenin signaling pathway is in part responsible for BBR-induced osteogenic differentiation of MSCs in vitro. BBR is a potential pharmaceutical medicine by enhancing osteogenic differentiation for bone disorders, such as osteoporosis.

Inhibitory effects of autologous γ-irradiated cell conditioned medium on osteoblasts in vitro.

Skeletal complications from radiation therapy have been reported in patients with breast, brain and pelvic cancer, and types of blood cancer. However, it remains to be elucidated whether localized radiotherapy may result in systemic adverse effects on the unirradiated skeleton through an abscopal mechanism. The present study investigated the abscopal effect of radiation on osteoblasts mediated by autologous γ-irradiated cell conditioned medium. Osteoblasts obtained from calvarial bones were incubated with irradiated cell conditioned medium (ICCM) and changes in cell viability, alkaline phosphatase (ALP) activity, mineralization ability, cell apoptosis and the gene expression levels of ALP, osteocalcin (BGP), osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL) and caspase 3 were observed. Notably, ICCM regulated osteoblast function, inhibiting viability and differentiation, resulting in apoptosis or cell death. ICCM at 10 or 20%, from osteoblasts irradiated with 10 Gy γ-rays, significantly inhibited the proliferation of osteoblastic cells (P<0.001). In addition, an increase in apoptosis was noted in the osteoblasts incubated with ICCM at 40% with increasing doses of radiation, accompanied by an upregulation in the mRNA expression of caspase 3. In addition, ICCM at 20% inhibited the ALP activity in the 5 and 10 Gy groups and osteoblast mineralization, particularly at 10 Gy ICCM. Additionally, the mRNA expression levels of ALP, BGP, OPG and RANKL of the cells treated with ICCM at 20% were downregulated significantly compared with those treated with medium from unirradiated cells. The present study provided novel evidence to elucidate radiation-therapy-associated side effects on the skeleton.

Rho-kinase limits BMP-4-stimulated osteocalcin synthesis in osteoblasts: regulation of the p38 MAP kinase pathway.

AIM: We previously reported that bone morphogenetic protein-4 (BMP-4) stimulates the synthesis of osteocalcin via p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells, whereas p44/p42 MAP kinase plays as a negative regulator in the synthesis. In the present study, we investigated whether Rho-kinase is involved in BMP-4-stimulated osteocalcin synthesis in MC3T3-E1 cells. MAIN METHODS: The levels of osteocalcin were measured by ELISA. The phosphorylation of each protein kinase was analyzed by Western blotting. The mRNA levels of osteocalcin were determined by real-time RT-PCR. KEY FINDINGS: BMP-4 induced the phosphorylation of myosin phosphatase targeting subunit-1 (MYPT-1), a substrate of Rho-kinase. Y27632 or fasudil, specific inhibitors of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly amplified the BMP-4-stimulated osteocalcin synthesis in a dose-dependent manner. The osteocalcin mRNA expression levels induced by BMP-4 were enhanced by Y27632 or fasudil. BMP-4-stimulated osteocalcin release was significantly up-regulated in Rho-knocked down cells with Rho A-siRNA. Y27632 or fasudil failed to affect the BMP-4-induced phosphorylation of SMAD1 or p44/p42 MAP kinase. On the other hand, Y27632 or fasudil markedly strengthened the phosphorylation levels of p38 MAP kinase induced by BMP-4. SIGNIFICANCE: These results strongly suggest that Rho-kinase negatively regulates BMP-4-stimulated osteocalcin synthesis via the p38 MAP kinase pathway in osteoblasts.

Effects of enamel matrix proteins on proliferation, differentiation and attachment of human alveolar osteoblasts.

OBJECTIVES: Enamel matrix proteins (EMPs) have been demonstrated to promote periodontal regeneration. However, effects of EMPs on human alveolar osteoblasts (hAOBs), up to now, have still been unclear. The purpose of this study was to investigate influence of EMPs on proliferation, differentiation and attachment of hAOBs in vitro. MATERIALS AND METHODS: EMPs were extracted using the acetic acid method, hAOBs were obtained and cultured in vitro. Cell proliferation, alkaline phosphatase (ALP) activity, mRNA expression of osteogenic markers and cell attachment were measured in the absence and in the presence of EMPs (50, 100 and 200 µg/ml). RESULTS: EMPs increased proliferation of hAOBs; however, they inhibited ALP activity and mRNA expression of osteogenic markers (collagen I, ALP, runt-related protein 2, osteocalcin, bone sialoprotein and osteopontin). Meanwhile, EMPs hindered hAOBs' attachment. These effects occurred in EMPs concentration-dependent manner. CONCLUSIONS: These results indicate that EMPs may inhibit osteoblastic differentiation and attachment to prevent ankylosis and allow other cell types to regenerate periodontal tissues.

Vimentin inhibits ATF4-mediated osteocalcin transcription and osteoblast differentiation.

Activating transcription factor 4 (ATF4) is an osteoblast-enriched transcription factor that regulates osteocalcin transcription and osteoblast terminal differentiation. To identify functional partners of ATF4, we applied ROS17/2.8 osteoblast nuclear extracts and purified recombinant His-ATF4 onto a Ni(+) affinity matrix chromatography column. Vimentin was identified by liquid chromatography-mass spectrometry. Coimmunoprecipitation and pulldown assays revealed that vimentin interacted with ATF4 with its first leucine zipper domain. DNA cotransfection and gel retardation demonstrated that vimentin inhibited the transactivation activity of ATF4 on osteocalcin by preventing it to bind OSE1, the ATF4 binding site on the osteocalcin promoter. Northern hybridization revealed that vimentin was expressed at a high level in immature osteoblasts and a low level in fully differentiated osteoblasts. Down-regulation of vimentin by small interfering RNA induced endogenous osteocalcin transcription in immature osteoblasts. Conversely, ectopic overexpression of vimentin in osteoblasts inhibited osteoblast differentiation as shown by lower alkaline phosphatase activity, delayed mineralization, and decreased expression of osteoblast marker genes such as bone sialoprotein and osteocalcin. Together, our data uncover a novel mechanism whereby a cytoskeletal protein, vimentin, acts as a break on differentiation in immature osteoblasts by interacting with ATF4.

Bone morphogenetic proteins are involved in the pathobiology of synovial chondromatosis.

Synovial chondromatosis is characterized by the formation of osteocartilaginous nodules (free bodies) under the surface of the synovial membrane in joints. Free bodies and synovium isolated from synovial chondromatosis patients expressed high levels of BMP-2 and BMP-4 mRNAs. BMP-2 stimulated the expression of Sox9, Col2a1, and Aggrecan mRNAs in free-body and synovial cells and that of Runx2, Col1a1, and Osteocalcin mRNAs in the synovial [corrected] cells only. BMP-2 increased the number of alcian blue-positive colonies in the free-body cell culture but not in the synovial cell culture. Noggin suppressed the expression of Sox9, Col2a1, Aggrecan, and Runx2 mRNAs in both the free-body and synovial cells. Further, it inhibited Osteocalcin expression in the synovial cells. These results suggest that BMPs are involved in the pathobiology of cartilaginous and osteogenic metaplasia observed in synovial chondromatosis.

Local application of statin promotes bone repair through the suppression of osteoclasts and the enhancement of osteoblasts at bone-healing sites in rats.

OBJECTIVE: We investigated whether the local administration of simvastatin affected both the cellular events and the bone formation at surgically created bone defects in rat. STUDY DESIGN: Simvastatin (or a vehicle) was injected into a rat bony defect for 3 consecutive days from the day of surgery. Five or ten days after the injection, new bone tissue was collected, and the gene expressions of bone-related proteins were examined. For the histomorphometry, new bone area was measured. RESULTS: At day 5, the statin group demonstrated significantly larger new bone area. The number of tartrate-resistant acid phosphatase-positive multinucleated cells in the statin group was less than in the control group. In the statin group, the expressions of both alkaline phosphatase and bone morphogenetic protein 2 mRNA significantly increased. In contrast, the expression of cathepsin K was significantly suppressed in the statin group. Although the levels of both RANK and osteoprotegerin were not affected by statin, the expression of RANKL was depressed. At day 10, there were no significant differences among the groups in either histomorphometric or reverse-transcription polymerase chain reaction analyses. CONCLUSION: New bone area increased under the influence of simvastatin; however, the effect did not continue when the administration was terminated. Osteoclast suppression may be the consequence of RANKL depression.

Mitogen activated protein kinase-dependent inhibition of osteocalcin gene expression by transforming growth factor-beta1.

TGF-beta (transforming growth factor-beta) plays a key role in osteoblast differentiation and bone development. While the ability of TGF-beta to inhibit the expression of osteoblast differentiation genes has been well documented, the mechanism of this inhibition is not yet completely characterized. Runx2, a transcription factor necessary for expression of osteoblast differentiation genes is a central target of inhibition by TGF-beta. In this study, we found that TGF-beta1 inhibits expression of osteoblast differentiation genes without altering expression of Runx2. Transient transfection experiments determined that TGF-beta1 inhibited osteocalcin promoter activity and this effect is mediated through Runx2. We further identified that there was no change in protein expression, cellular localization, or DNA binding affinity of Runx2 after TGF-beta1-treatment of osteoblasts, suggesting that Runx2 undergoes post-translational modifications following TGF-beta1 treatment. Co-immunoprecipitation experiments identified increased phosphorylation of Runx2 when differentiating osteoblasts were treated with TGF-beta1. Mitogen activated protein kinase (MAPK) inhibitors relieved the TGF-beta1-inhibitory effect of Runx2-mediated osteocalcin expression. Thus, our results suggest that TGF-beta1-inhibition of osteoblast differentiation is dependent on the MAPK pathway and this effect is most likely mediated by post-translational modification of Runx2 such as phosphorylation rather than other regulatory mechanisms.

The sympathetic tone mediates leptin's inhibition of insulin secretion by modulating osteocalcin bioactivity.

The osteoblast-secreted molecule osteocalcin favors insulin secretion, but how this function is regulated in vivo by extracellular signals is for now unknown. In this study, we show that leptin, which instead inhibits insulin secretion, partly uses the sympathetic nervous system to fulfill this function. Remarkably, for our purpose, an osteoblast-specific ablation of sympathetic signaling results in a leptin-dependent hyperinsulinemia. In osteoblasts, sympathetic tone stimulates expression of Esp, a gene inhibiting the activity of osteocalcin, which is an insulin secretagogue. Accordingly, Esp inactivation doubles hyperinsulinemia and delays glucose intolerance in ob/ob mice, whereas Osteocalcin inactivation halves their hyperinsulinemia. By showing that leptin inhibits insulin secretion by decreasing osteocalcin bioactivity, this study illustrates the importance of the relationship existing between fat and skeleton for the regulation of glucose homeostasis.

Inhibitory effect of enamel matrix derivative on osteoblastic differentiation of rat calvaria cells in culture.

BACKGROUND AND OBJECTIVE: The effect of enamel matrix derivative (EMD) on bone differentiation remains unclear. Transforming growth factor beta1 (TGF-beta1) is reported to be contained in EMD. The aim of this study was to clarify the effect of EMD on osteoblastic cell differentiation and the possible role of TGF-beta1. MATERIAL AND METHODS: Fetal rat carvarial cells were treated with 10, 50 or 100 microg/ml EMD for 5-17 days. Alkaline phosphatase (ALP) activity and bone nodule formation were measured, and mRNA expressions of bone matrix proteins and core binding factor were analysed. RESULTS: Enamel matrix derivative inhibited ALP activity from the early stage of culture (29-44% inhibition) on days 5 and 10 and decreased bone nodule formation by 37-67% on day 17. These effects of EMD were concentration dependent. Enamel matrix derivative inhibited mRNA expression of osteocalcin and core binding factor. A high level of the active form of TGF-beta1 protein was detected in the conditioned medium treated with 100 microg/ml EMD. Treatment with TGF-beta1 antibody partly restored the inhibitory effect of EMD on ALP activity. CONCLUSION: Enamel matrix derivative inhibited the osteoblastic differentiation of rat carvarial cells and this was partly mediated by an increase in the activated form of TGF-beta1, suggesting that EMD may function initially to inhibit osteoblastic differentiation to allow a predominant formation of other periodontal tissues.

BGLAP is expressed in pancreatic cancer cells and increases their growth and invasion.

BACKGROUND: Bone gamma-carboxyglutamate protein (BGLAP; osteocalcin) is a small, highly conserved molecule first identified in the mineralized matrix of bone. It has been implicated in the pathophysiology of various malignancies. In this study, we analyzed the expression and role of BGLAP in the normal human pancreas, chronic pancreatitis (CP), and pancreatic ductal adenocarcinoma (PDAC) using quantitative RT-PCR, immunohistochemistry, immunocytochemistry and enzyme immunoassays, as well as cell proliferation and invasion assays. Gene silencing was carried out using specific siRNA molecules. RESULTS: Compared to the normal pancreas, BGLAP mRNA and protein levels were not significantly different in CP and PDAC tissues. BGLAP was faintly present in the cytoplasm of normal acinar cells but was strongly expressed in the cytoplasm and nuclei of tubular complexes and PanIN lesions of CP and PDAC tissues. Furthermore, BGLAP expression was found in the cancer cells in PDAC tissues as well as in 4 cultured pancreatic cancer cell lines. TNFalpha reduced BGLAP mRNA and protein expression levels in pancreatic cancer cell lines. In addition, BGLAP silencing led to reduction of both cell growth and invasion in those cells. CONCLUSION: BGLAP is expressed in pancreatic cancer cells, where it potentially increases pancreatic cancer cell growth and invasion through autocrine and/or paracrine mechanisms.

Osteogenic differentiation induced by bone morphogenetic proteins can be suppressed by platelet-released supernatant in vitro.

Both bone morphogenetic proteins (BMPs) and growth factors released from activated platelets both occur at sites of bone regeneration but their functional relationship to regulate the temporal and spatial sequence of cellular events is not well defined. Here we investigated whether supernatants derived from activated platelets can modulate the response of the osteogenic cell line MC3T3-E1 to BMPs, and whether BMPs have an effect on MC3T3-E1 cells stimulated with platelet-released supernatant. Platelet-released supernatant suppressed BMP-2-, BMP-6-, and BMP-7-induced osteogenic differentiation of MC3T3-E1 cells, as indicated by the significant decrease in alkaline phosphatase activity and lower levels of osteocalcin transcripts, whereas BMP-2, BMP-6, and BMP-7 did not modulate migration and proliferation of MC3T3-E1 cells, which were stimulated with platelet-released supernatant. Osteogenic differentiation in response to BMPs was not affected after precultivation of MC3T3-E1 cells with platelet-released supernatant. These data suggest that activated platelets can provide a microenvironment that temporarily suppresses the differentiation of osteogenic cells in the presence of BMPs. Future strategies to stimulate bone regeneration should take the suppression of BMP-induced osteogenic differentiation during the existence of the blood clot into account.

Activation of peroxisome proliferator-activated receptor-gamma inhibits the Runx2-mediated transcription of osteocalcin in osteoblasts.

Mesenchymal cells are able to differentiate into several distinct cell types, including osteoblasts and adipocytes. The commitment to a particular lineage may be regulated by specific transcription factors. Peroxisome proliferator-activated receptor-gamma (PPARgamma), acting in conjunction with CCAAT/enhancer-binding protein-alpha, has been suggested as a key regulator of adipogenic differentiation. Previous studies have shown that the activation of PPARgamma in osteoblasts suppresses osteoblast differentiation and the expression of osteocalcin, an osteoblast-specific protein. However, the mechanism of this inhibition remains unclear. We investigated the effect of PPARgamma activation on the expression of osteocalcin and analyzed the molecular mechanism. Mouse osteoblastic MC3T3-E1 cells expressed PPARgamma, which was transcriptionally active, whereas rat osteosarcoma ROS 17/2.8 cells did not. Treatment of MC3T3-E1 osteoblasts and ROS 17/2.8 cells stably transfected with PPARgamma2 with the PPARgamma activator 15-deoxy-Delta12,14-prostaglandin J2 inhibited the mRNA expression of osteocalcin and Runx2, the latter of which is a key transcription factor in osteoblast differentiation. This decreased expression of osteocalcin and Runx2 was partly explained by the decreased level of Runx2 resulting from the suppressed transcription from the Runx2 promoter. However, in addition to this indirect effect, the activation of PPARgamma by 15-deoxy-Delta12,14-prostaglandin J2 directly suppressed the Runx2-mediated induction of the activities of the osteocalcin promoter and the artificial promoter p6OSE2, which contains six tandem copies of osteoblast-specific element-2, the Runx2-binding promoter sequence. This inhibition was mediated by a physical interaction between PPARgamma and Runx2 and the subsequent repression of the transcriptional activity at the osteoblast-specific element-2 sequence. Thus, this study demonstrates that the activation of PPARgamma inhibits osteocalcin expression both by suppressing the expression of Runx2 and by interfering with the transactivation ability of Runx2.

Activation of p38 mitogen-activated protein kinase is required for osteoblast differentiation.

p38 MAPK is a conserved subfamily of MAPKs involved in inflammatory response, stress response, cell growth and survival, as well as differentiation of a variety of cell types. In this report we demonstrated that p38 MAPK played an important role in osteoblast differentiation using primary calvarial osteoblast, bone marrow osteoprecursor culture, and a murine cell line, MC3T3-E1. We found that p38 MAPK was activated as calvarial osteoblast differentiates along with extracellular signal-regulated kinases (ERKs). When p38 MAPK is inhibited with a specific inhibitor, the expression of differentiation markers, such as alkaline phosphatase and mineral deposition, were significantly reduced. MC3T3-E1 cells expressing dominant negative p38 MAPK also displayed signs of delay in ALP and mineral deposition. Differentiation of the bone marrow osteoprecursors was also impeded by the p38 MAPK inhibitor, justified by the same markers. Yet the inhibitory effects observed in calvarial osteoblasts and bone marrow osteoprogenitor cells could be partially prevailed by bone morphogenetic protein-2. Inhibition of ERKs with a specific drug did not significantly affect osteoblast differentiation even though ERK1/2 were also activated during osteoblast differentiation. These results taken together indicate that p38 MAPK, but not ERKs, is necessary for osteoblast differentiation.

Effect of estrogen and 1alpha,25(OH)2- vitamin D3 on the activity and growth of human primary osteoblast-like cells in vitro.

OBJECTIVE: To evaluate effects of 17beta-E(2) and 1alpha,25(OH)(2)-vitamin D(3) on human osteoblast-like (hOB) cells. DESIGN: Controlled, experimental study. SETTING: University hospital. PATIENT(S): hOB cell cultures were prepared from the upper femur of postmenopausal patients undergoing bipolar endoprosthesis arthroplasty for a fractured femoral neck. INTERVENTION(S): hOB cells were subcultured with either 17beta-E(2) or 1alpha,25(OH)(2)-vitamin D(3), or both. MAIN OUTCOME MEASURE(S): Cell proliferation and activity of alkaline phosphatase, osteocalcin, and interleukin-6. RESULT(S): 17beta-E(2) significantly reduced interleukin-6 and osteocalcin to 34% and 60% of control value but induced alkaline phosphatase and cell proliferation to 183% and 150% of control value. 1alpha,25(OH)(2)-vitamin D(3) significantly decreased cell proliferation to 88% of that of control group, but 1alpha,25(OH)(2)-vitamin D(3) plus 17beta-E(2) showed no difference from the control group. Alkaline phosphatase and osteocalcin were significantly increased by 1alpha,25(OH)(2)-vitamin D(3) alone or combined with 17beta-E(2), to 169% and 198% and to 144% and 144% of control value, respectively. 1alpha,25(OH)(2)-vitamin D(3), with or without 17beta-E(2), decreased interleukin-6 levels to 27% and 38% of control group, respectively. CONCLUSION(S): 17beta-E(2) and 1alpha,25(OH)(2)-vitamin D(3) have effects on osteoblasts. The prevention of osteoporosis by estrogen may be related not only to direct effects on osteoblastic activity and proliferation but also to indirect effects on osteoclasts by the decrease of interleukin-6 secretion.

Additive effects of raloxifene and alendronate on bone density and biochemical markers of bone remodeling in postmenopausal women with osteoporosis.

Both raloxifene (RLX) and alendronate (ALN) can treat and prevent new vertebral fractures, increase bone mineral density (BMD), and decrease biochemical markers of bone turnover in postmenopausal women with osteoporosis. This phase 3, randomized, double-blind 1-yr study assessed the effects of combined RLX and ALN in 331 postmenopausal women with osteoporosis (femoral neck BMD T-score, less than -2). Women (aged < or = 75 yr; > or = 2 yr since their last menstrual period) received placebo, RLX 60 mg/d, ALN 10 mg/d, or RLX 60 mg/d and ALN 10 mg/d combined. At baseline, 6 and 12 months, BMD was measured by dual x-ray absorptiometry. The bone turnover markers serum osteocalcin, bone-specific alkaline phosphatase, and urinary N- and C-telopeptide corrected for creatinine were measured. The effects of RLX and ALN were considered to be independent and additive if the interaction effect was not statistically significant (P > 0.10) in a two-way ANOVA model. All changes in BMD and bone markers at 12 months were different between placebo and each of the active treatment groups, and between the RLX and RLX+ALN groups (P < 0.05). On average, lumbar spine BMD increased by 2.1, 4.3, and 5.3% from baseline with RLX, ALN, and RLX+ALN, respectively. The increase in femoral neck BMD in the RLX+ALN group (3.7%) was greater than the 2.7 and 1.7% increases in the ALN (P = 0.02) and RLX (P < 0.001) groups, respectively. The changes from baseline to 12 months in bone markers ranged from 7.1 to -16.0% with placebo, -23.8 to -46.5% with RLX, -42.3 to -74.2% with ALN, and -54.1 to -81.0% in the RLX+ALN group. RLX and ALN increased lumbar spine and femoral neck BMD, and decreased osteocalcin and C-telopeptide corrected for creatinine in an additive and independent manner, because the interaction effects were not significant. Although the ALN group had changes in BMD and bone markers that were approximately twice the magnitude as in the RLX group, it is not known how well these changes correlate to the clinical outcome of fracture. RLX+ALN reduced bone turnover more than either drug alone, resulting in greater BMD increment, but whether this difference reflects better fracture risk reduction was not assessed in this study.

Protein kinase C does not mediate the inhibitory action of lead on vitamin D3-dependent production of osteocalcin in osteoblastic bone cells.

The level of osteocalcin in serum is lower in lead-intoxicated children than in their normal counterparts. To explain this clinical observation, we investigated the mechanism of action of lead on vitamin D3-dependent osteocalcin production. Lead (5-20 microM) blocked the stimulating effects of vitamin D3 on osteocalcin production in cultured rat osteosarcoma cells (ROS 17/2.8). It is often suggested that activation of protein kinase C (PKC) is a critical mediator of the toxic actions of lead. Treatment of ROS cells with Gö6976, an inhibitor of PKC alpha and beta isozymes, produced similar effects as lead on vitamin D3-dependent osteocalcin production, while activation of PKC by phorbol-12-myristate-13-acetate (TPA) did not reverse or mimic this effect of lead. Thus activation of PKC is not consistent with the actions of lead on vitamin D3-dependent osteocalcin production. Measurement of PKC enzyme activity showed that 10 microM lead treatment does not activate or inhibit the activity of PKC in ROS cells. Western blot analysis indicated that lead treatment does not translocate PKC alpha, beta, or zeta from cytosol to membrane. Therefore, we concluded that PKC does not mediate the cellular toxicity of lead on vitamin D3-dependent osteocalcin production.