Rat Osteoblastic Cell Line Research Articles

Anti-osteoporotic activities of the collagen extracted from Carapacis et Plastri Testudinis on a ROS1728 cell line in vitro

Purpose: The decoction of collagen extracted from Carapacis et Plastri Testudinis (CCPT) has been reported to have an effect of anti-osteoporosis. The present study was to investigate the in vitro molecular mechanisms effect of CCPT on ROS1728 differentiation and proliferation. Methods: Rat osteoblast cell line (ROS 1728) was cultured and exposed to CCPT at different concentrations. The effect of CCPT on the proliferation of ROS1728 was evaluated through detecting the activity of Alkaline Phosphatase (ALP) and the type I collagen (Coll-I) content. The gene expression of ALP, Coll-I, Osteocalcin (OC), Runx2, Osterix (Osx), Osteoprotegerin (OPG) and RANKL (receptor activator of NF-MB ligand) was observed using RT-PCR analysis. Results: The results showed that CCPT treatment (2.5, 5.0 and 10.0 mg/ml) affected the proliferation of ROS1728 in a concentration-dependent manner. The highest expression was obtained by treatment with 5 mg/ml of CCPT after a nine-day treatment duration based on ALP activity and Coll-I content (P<0.01) with the control group, whereas, the expression of ALP, Coll-I, OC, Runx2, Osx and OPG mRNA was facilitated (P<0.01). Only the level of RANKL mRNA decreased compared with the control group (P<0.01). Conclusion: Use of CCPT can induce ROS1728 proliferation into osteoblasts and enhance the function of osteogenesis through regulating the gene expression of Runx2 and the osteogenesis-specific transcription factors of Osx, and inhibit bone absorption by OPG/RANKL/RANK signaling pathways.

LncRNA-H19 Modulates Wnt/β-catenin Signaling by Targeting Dkk4 in Hindlimb Unloaded Rat.

To investigate the biological functions of long noncoding RNA-H19 (H19) in the pathogenesis of disuse osteoporosis (DOP). Fifty-four male Sprague Dawley (SD) rats were randomly divided into three groups: baseline control (BC, 6), age-matched control (AC, 24), and hindlimb unloading (HLU, 24). The rats in the BC group were sacrificed at the beginning of the experiment, while the AC and HLU rats were sacrificed at different times (7, 14, 21 and 28 days after HLU). The DOP model was verified by micro-CT scan, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to quantify the expression of osteogenic genes (OPG, RunX2 and OPG). Gene sequencing and bioinformatic analysis were performed to find H19 target genes and the associated signaling pathway, which were first verified on tissue samples. Further verification was performed by knocking down the H19 and related gene in rat osteoblast cell line (UMR106 cell). Then, the changes of associated signaling pathway and osteogenic function were examined to confirm the prediction of the bioinformatic analysis. Micro-CT scans and quantitative real-time polymerase chain reaction (qRT-PCR) tests showed progressively deteriorated trabecular bone and decreased level of osteogenic genes in the metaphysis of distal femur during HLU, indicating the successful establishment of a DOP model. According to RNA sequencing, 1351 mRNA and 464 lncRNA were abnormally expressed in response to mechanical unloading, in which the H19 decreased 2.86 fold in HLU rats. There were 1426 mRNA predicted to be the target genes of H19, and KEGG pathway analysis suggested that Wnt signaling pathway (Wnt signaling) was the top pathway responsible for these target genes. In the Wnt-associated genes targeted by H19, 11 were differentially expressed between HLU and AC rats, among which Dkk4 increased 2.44 fold in HLU rats when compared to normal controls. These results of sequencing and bioinformatic analysis were confirmed by the low expression of H19, overexpression of Dkk4 and inhibited Wnt signaling observed in DOP rats. Subsequent in vitro cell assay further demonstrated that knockdown of H19 led to upregulation of Dkk4, and inhibition of Wnt signaling and osteogenic function in UMR106 cell. These effects can be greatly reversed after application of knocking down Dkk4. Our findings demonstrated that low expression of H19, induced by mechanical unloading, leads to development of DOP through inhibition of Wnt signaling by promoting Dkk4 expression.

Phosphate enhances Fgf23 expression through reactive oxygen species in UMR-106 cells.

Fibroblast growth factor 23 (FGF23) has been shown to work as a phosphotropic hormone. Although FGF23 reduces the serum phosphate level, it has not been established that phosphate directly regulates FGF23 production. In this study, we investigated whether phosphate can enhance Fgf23 expression using the rat osteoblastic cell line UMR-106, which has been shown to express Fgf23 in response to 1,25-dihydroxyvitamin D [1,25(OH)2D]. Phosphate increased Fgf23 expression in a dose- and time-dependent manner in the presence of 1,25(OH)2D. Phosphate also increased Fgf23 promoter activity, but showed no effect on the half-life of Fgf23 messenger RNA. Phosphonoformic acid and PD98059, an inhibitor of MEK, inhibited the effects of phosphate on Fgf23 expression and promoter activity. In addition, phosphate enhanced production of reactive oxygen species (ROS) in UMR-106 cells, and hydrogen peroxide enhanced FGF23 production in a dose- and time-dependent manner. Hydrogen peroxide also enhanced Elk1 reporter activity, a target of the MEK-extracellular-signal-regulated kinase (ERK) pathway. Furthermore, the effect of phosphate on ROS production and Fgf23 expression was inhibited by apocynin, an inhibitor of NADPH oxidase. These results indicate that phosphate directly enhances Fgf23 transcription without affecting the stability of Fgf23 messenger RNA by stimulating NADPH-induced ROS production and the MEK-ERK pathway in UMR-106 cells.

Parathyroid Hormone Regulates Histone Deacetylase (HDAC) 4 through Protein Kinase A-mediated Phosphorylation and Dephosphorylation in Osteoblastic Cells

Histone deacetylases (HDACs) are crucial regulators of gene expression in transcriptional co-repressor complexes. Previously, we reported that HDAC4 was a basal repressor of matrix metalloproteinase-13 (MMP-13) transcription and parathyroid hormone (PTH) regulates HDAC4 to control MMP-13 promoter activity through dissociation from Runx2. Here, we show that PTH induces the protein kinase A (PKA)-dependent phosphorylation of HDAC4 in the nucleus of the rat osteoblastic cell line, UMR 106-01. We demonstrate that PKA-dependent phosphorylated HDAC4 is released from Runx2 bound to the MMP-13 promoter in these cells. Point mutation of Ser-740 in rHDAC4 prevents the release of HDAC4 from Runx2 on the MMP-13 promoter and also prevents the PTH stimulation of MMP-13 transcription. Thus, PTH-induced phosphorylation of rHDAC4 at Ser-740 is crucial for regulating MMP-13 transcription in osteoblasts. PTH causes degradation of HDAC4, and this product appears in the cytoplasm. The cytoplasmic degradation of HDAC4 is blocked by PKA and lysosomal inhibitors, but is not affected by proteasome, caspase-3, or serine and aspartic protease inhibitors. In addition, the phosphatase inhibitor, okadaic acid, prevents degradation indicating that dephosphorylation is associated with degradation. These mechanisms regulating HDAC4 and their roles in such processes are crucial for bone and chondrocyte development. Our data support a link between PTH regulating HDAC4 phosphorylation by PKA, trafficking, partial degradation, and the control of MMP-13 transcription through association with Runx2.

HDAC4 Represses Matrix Metalloproteinase-13 Transcription in Osteoblastic Cells, and Parathyroid Hormone Controls This Repression

Parathyroid hormone (PTH) is a hormone regulating bone remodeling through its actions on both bone formation and bone resorption. Previously we reported that PTH induces matrix metalloproteinase-13 (MMP-13) transcription in osteoblastic cells. Here, we show that histone deacetylase 4 (HDAC4) interacts with Runx2, binds the MMP-13 promoter, and suppresses MMP-13 gene transcription in the rat osteoblastic cell line, UMR 106-01. PTH induces the rapid cAMP-dependent protein kinase-dependent release of HDAC4 from the MMP-13 promoter and subsequent transcription of MMP-13. Knock-out of HDAC4 either by siRNA in vitro or by gene deletion in vivo leads to an increase in MMP-13 expression, and overexpression of HDAC4 decreases the PTH induction of MMP-13. All of these observations indicate that HDAC4 represses MMP-13 gene transcription in bone. Moreover, PTH stimulates HDAC4 gene expression and enzymatic activity at times corresponding to the reassociation of HDAC4 with the MMP-13 promoter and a decline in its transcription. Thus, HDAC4 is a basal repressor of MMP-13 transcription, and PTH regulates HDAC4 to control MMP-13 promoter activity. These data identify a novel and discrete mechanism of regulating HDAC4 levels and, subsequently, gene expression.

Chloride intracellular channel 1 regulates osteoblast differentiation

We have identified chloride intracellular channel 1 (CLIC1) through proteomic approach, which was increased in response to canonical wnt signaling while being almost shut-off by adipogenic treatment in mouse mesenchymal C3H10T1/2 cells. We found that CLIC1 was expressed in mouse (MC3T3-E1), rat (ROS 17/2.8 and UMR-106) or human (MG63 and SaOS2) osteoblastic cell lines as well as primary culture of mouse calvarial cells by RT-PCR or Western blot analysis. The expression level of CLIC1 is increased upon treatment of osteogenic medium, whereas it almost disappeared in adipogenic condition, confirming the proteomic data. The expression of CLIC1 was localized mainly in nuclear membrane and vesiculo-cytoplasmic, the latter of which was colocalized with mitochondria. Retroviral overexpression of CLIC1 did not increase whole-cell current but induces hyperpolarization of mitochondrial membrane potential estimated using the fluorescent dye TMRE. Moreover, overexpression of CLIC1 resulted in increase in osteoblastic differentiation of C3H10T1/2 cells as measured by ALP activities or osteoblastic gene expression (osterix, ALP and osteocalcin), although it did not result in induction of Runx2 transcription activities at mouse osteocalcin (OG2) promoter. Finally, in vitro knock-down of CLIC1 using stable siRNA CLIC1 significantly suppressed osteoblastic differentiation. Taken together, these results suggest that CLIC1 may play a role in the regulation of osteoblastic differentiation from mesenchymal progenitors, although its physiologic role in osteoblasts remains to be determined.

Nitric oxide promotes the progression of periapical lesion via inducing macrophage and osteoblast apoptosis

This study aimed to elucidate the modulation by nitric oxide (NO) of the apoptosis of macrophages and osteoblasts, the essential cellular components in the development of periapical lesions. Lipopolysaccharide (LPS) induced prominent nitrite synthesis in J774 mouse macrophage cell lines. Exposure to LPS induced obvious apoptosis in J774 cells, whereas transient transfection with murine inducible nitric oxide synthase (iNOS), small interfering RNA (siRNA) diminished this effect. Tumor necrosis factor-alpha (TNF-alpha) and S-nitroso-N-acetyl-DL-penicillamine (SNAP) (a NO donor) triggered apoptosis in UMR-106 rat osteoblastic cell lines and a synergistic effect was noted when TNF-alpha and SNAP were added to the medium together. Administration of siRNAs for c-Fos and c-Jun: components of activator protein-1 (AP-1) and transforming growth factor-beta1 attenuated the combined effect markedly. Terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) stain in a rat model of induced periapical lesion showed positive apoptotic signals in macrophages and osteoblasts. Administration of N(G)-monomethyl-l-arginine markedly diminished the extent of bone loss and the amounts of apoptotic macrophages and osteoblasts. In conclusion, NO mediates LPS-stimulated apoptosis of macrophages. It also induces osteoblast apoptosis and augments the pro-apoptotic effect of cytokines. Inhibition of NO synthesis in vivo attenuates apoptosis and the size of periapical lesions. Taken together, these results suggest that NO may promote the progression of periapical lesion by inducing the apoptosis of macrophages and osteoblasts.

Cyclosporine-Induced Apoptosis in Osteosarcoma Cells

Introduction Posttransplant bone disease is one of the complications of cyclosporine (CsA), which is widely used as an immunosuppressive agent in the field of kidney transplantation. Cyclosporine treatment causes osteopenia as a result of altered bone turnover, but the pathogenic mechanisms of this process remain unclear. This study examined the ability of CsA to induce apoptosis in a rat osteoblast cell line. Results We induced apoptosis in rat osteoblastic ROS 17/2.8 cells by exposure to CsA. MTT assay showed that CsA exhibited significant cytotoxic effects on ROS 17/2.8 cells in a dose-dependent manner. Western blot analysis showed enhanced processing of caspase-8, Bax, and p53 after CsA treatment. Expression of cleaved poly (ADP-ribose) polymerase (PARP) was elevated by CsA treatment. Pro-caspase-3 and Bcl-2 proteins were decreased by CsA. Conclusions These results suggested that CsA induced apoptosis of osteoblasts.

ASBMR 27th Annual Meeting 1001–1228

We hypothesized that by taking subjects from the extremes of a population, we would specifically find alleles related to the bone mineral density trait, if such alleles exist for any particular target gene. We tested this idea using RUNX2, the well known osteoblast transcription factor. From a population of 1300 subjects the age-weight adjusted femoral neck bone mineral density (BMD) was ranked and the upper and lower deciles taken to represent the adjusted extremes. After adjusting and ranking, the two groups (n=130 each) were not significantly different for age or weight. In these 260 subjects, we identified 19 allelic variations within the RUNX2 gene and promoter (P1 and P2), and characterized these novel variations with respect to strata by genotype using dHPLC. A general linkage disequilibrium (LD) analysis of RUNX2 showed that LD drops off with physical distance, as expected. Within the P2 promoter were three polymorphic nucleotides for which the minor alleles were over represented in the upper decile of BMD. These alleles are in complete LD with each other and represent a haplotype block that is significantly associated with increased BMD. Constructs made from the and the BMD alleles were cloned upstream of otherwise identical luciferase constructs. Despite there being only 3 nucleotide differences between the two constructs, when transfected into osteoblast-like cells the high allele construct showed significantly greater P2 promoter activity than the normal allele. Comparative homology studies showed that the high alleles were the major forms in other animals (dog, rat, mouse, chimp), suggesting that the human normal alleles represent the evolutionary change. The rat sequence was identical to the human high allele, permitting an analysis of DNAprotein interactions in the well characterized ROS17/2.8 rat osteoblast cell line. These data showed abundant nuclear factor binding to one the three polymorphic sites in the human normal allele that does not occur in the high allele (and rat sequence). The data suggest that the high allele does not interact with a factor that otherwise binds strongly to the normal human allele. Since the high allele had higher P2 promoter activity, the binding activity may be a repressor that limits the expression of the normal allele. In any event, the nuclear factor is a candidate for the allele-specific regulation of the RUNX2 promoter.

Osteoblast interactions with calcium phosphate ceramics modified by coating with type I collagen

Complications associated with the use of autogenous bone in the repair or replacement of tissue lost through injury or disease have driven the search for alternative sources of graft material. Bioceramics containing hydroxyapatite (HA), tricalcium phosphate (TCP), or composites that combine the best properties of both of these materials are among the principal candidates. In this study, we have investigated the in vitro proliferation, morphology, and viability of an immortalized rat osteoblast cell line cultured on HA, TCP, and composites of the two in the ratios 75:25 (H75), 50:50 (H50), and 25:75 (H25) for 28 days. The biocompatibility of each material was examined in the presence and absence of a collagen coating. With the exception of H50, cell proliferation, quantified by carboxyfluorescein fluorescence, was enhanced by collagen coating of all materials for the first 14 days, although at later time points cell numbers were unaffected. It is notable that the collagen coating was least stable on H50, the only material not to show enhancement of cell growth on coating. Confocal laser scanning microscopy confirmed that cell growth was more extensive on coated materials over the first 7-14 days in culture, and the development of cell extensions and bridges across the pores in the materials was observed. Results indicate that collagen coating of calcium phosphate ceramics may also increase their compatibility and osseointegration in vivo.

Parathyroid hormone stimulation and PKA signaling of latent transforming growth factor‐β binding protein‐1 (LTBP‐1) mRNA expression in osteoblastic cells

Parathyroid hormone (PTH) regulates bone remodeling and calcium homeostasis by acting on osteoblasts. Recently, the gene expression profile changes in the rat PTH (1-34, 10(-8)M)-treated rat osteoblastic osteosarcoma cell line, UMR 106-01, using DNA microarray analysis showed that mRNA for LTBP-1, a latent transforming growth factor (TGF-beta)-binding protein is stimulated by PTH. Latent TGF-beta binding proteins (LTBPs) are required for the proper folding and secretion of TGF-beta, thus modifying the activity of TGF-beta, which is a local factor necessary for bone remodeling. We show here by real time RT-PCR that PTH-stimulated LTBP-1 mRNA expression in rat and mouse preosteoblastic cells. PTH also stimulated LTBP-1 mRNA expression in all stages of rat primary osteoblastic cells but extended expression was found in differentiating osteoblasts. PTH also stimulated TGF-beta1 mRNA expression in rat primary osteoblastic cells, indicating a link between systemic and local factors for intracellular signaling in osteoblasts. An additive effect on LTBP-1 mRNA expression was found when UMR 106-01 cells were treated with PTH and TGF-beta1 together. We further examined the signaling pathways responsible for PTH-stimulated LTBP-1 and TGF-beta1 mRNA expression in UMR 106-01 cells. The PTH stimulation of LTBP-1 and TGF-beta1 mRNA expression was dependent on the PKA and the MAPK (MEK and p38 MAPK) pathways, respectively in these cells, suggesting that PTH mediates its effects on osteoblasts by several intracellular signaling pathways. Overall, we demonstrate here that PTH stimulates LTBP-1 mRNA expression in osteoblastic cells and this is PKA-dependent. This event may be important for PTH action via TGF-beta in bone remodeling.

Parathyroid Hormone Uses Multiple Mechanisms to Arrest the Cell Cycle Progression of Osteoblastic Cells from G1 to S Phase

Parathyroid hormone (PTH) plays a major role in bone remodeling and has the ability to increase bone mass if administered daily. In vitro, PTH inhibits the growth of osteoblastic cell lines, arresting them in G(1) phase. Here, we demonstrate that PTH regulates the expression of at least three genes to achieve the following: inducing expression of MAPK phosphatase 1 (MKP-1) and p21(Cip1) and decreasing expression of cyclin D1 at both mRNA and protein levels. The induction of MKP-1 causes the dephosphorylation of extracellular signal-regulated kinase and therefore the decrease in cyclin D1. Overexpression of MKP-1 arrests UMR cells in G(1) phase. The mechanisms involved in PTH regulation of these genes were studied. Most importantly, PTH administration produces similar effects on expression of these genes in rat femoral metaphyseal primary spongiosa. Analyses of p21(Cip1) expression levels in bone indicate that repeated daily PTH injections make the osteoblast more sensitive to successive PTH treatments, and this might be an important feature for the anabolic functions of PTH. In summary, our data suggest that one mechanism for PTH to exert its anabolic effect is to arrest the cell cycle progression of the osteoblast and hence increase its differentiation.

1,25-Dihydroxyvitamin D3 Down-regulation of PHEX Gene Expression Is Mediated by Apparent Repression of a 110 kDa Transfactor That Binds to a Polyadenine Element in the Promoter

The PHEX gene encodes an endopeptidase expressed in osteoblasts that inactivates an uncharacterized peptide hormone, phosphatonin, which suppresses bone mineralization as well as renal phosphate reabsorption and vitamin D bioactivation. We demonstrate that 1alpha-25-dihydroxyvitamin D (1,25(OH)2D3), the, active renal vitamin D metabolite, decreases PHEX mRNA in the rat osteoblastic cell line, UMR-106, as well as in mouse calvaria. Promoter/reporter construct analysis of the murine PHEX gene in transfected UMR-106 cells localized the repressive effect of 1,25(OH)2D3 to the -133 to -74 bp region, and gel mobility shift experiments revealed that 1,25(OH)2D3 treatment of the cells diminished the binding of a nuclear protein(s) to a stretch of 17 adenines from bp -116 to -100 in the proximal PHEX promoter. Either overexpression of a dominant-negative vitamin D receptor (VDR) or deletion of this sequence of 17 A-T base pairs abolished the repressive effect of 1,25(OH)2D3 by attenuating basal promoter activity, indicating that this region mediates the 1,25(OH)2D3 response and is involved in basal transcription. South-western blot analysis and DNA affinity purification show that an unidentified 110 kDa nuclear protein binds to the poly(A) element. Because 1,25(OH)2D3-liganded VDR neither binds to the polyadenine region of the PHEX promoter nor directly influences the association of the 110 kDa transfactor, we conclude that 1,25(OH)2D3 indirectly decreases PHEX expression via VDR-mediated repression (or modification) of this novel transactivator. Thus, we have identified a cis-element required for PHEX gene transcription that participates in negative feedback control of PHEX expression and thereby modulates the actions of phosphatonin.

Transforming Growth Factor-β1 Regulation of Collagenase-3 Expression in Osteoblastic Cells by Cross-talk between the Smad and MAPK Signaling Pathways and Their Components, Smad2 and Runx2

Transforming growth factor-beta (TGF-beta) plays a key role in osteoblast differentiation and bone development and remodeling. Collagenase-3 (matrix metalloproteinase-13) is expressed by osteoblasts and seems to be involved in osteoclastic bone resorption. Here, we show that TGF-beta 1 stimulates collagenase-3 expression in the rat osteoblastic cell line UMR 106-01 and requires de novo protein synthesis. Dominant-negative Smad2/3 constructs indicated that Smad signaling is essential for TGF-beta 1-stimulated collagenase-3 promoter activity. Inhibitors of the ERK1/2 and p38 MAPK pathways, but not the JNK pathway, reduced TGF-beta 1-stimulated collagenase-3 expression, indicating that the p38 MAPK and ERK1/2 pathways are also required for TGF-beta 1-stimulated collagenase-3 expression in UMR 106-01 cells. These inhibitors did not prevent nuclear localization of Smad proteins, but they inhibited Smad-mediated transcriptional activation. We have shown for the first time that Runx2 (a bone transcription factor and a potential substrate for the MAPK pathway) is phosphorylated in response to TGF-beta 1 treatment in osteoblastic cells. Cotransfection of Smad2 and Runx2 constructs had a cooperative effect on TGF-beta 1-stimulated collagenase-3 promoter activity in these cells. We further identified ligand-independent physical interaction between Smad2 and Runx2. Taken together, our results provide an important role for cross-talk between the Smad and MAPK pathways and their components in expression of collagenase-3 following TGF-beta 1 treatment in UMR 106-01 cells.

In vitro studies with the calcimimetic, cinacalcet HCl, on normal human adult osteoblastic and osteoclastic cells.

This study examined whether the calcium-sensing receptor (CaR) is expressed in normal adult human osteoblastic and osteoclastic cells in culture, and whether the calcimimetic, cinacalcet HCl (AMG 073), potentiates the effects of calcium (via CaR, or some other receptor/mechanism). When mouse or human osteoblastic cells were treated with higher concentrations of calcium (6.6 or 8.6 mM in alpha-MEM/10% FBS) than present in control cultures (1.6 mM), the previously well-documented increase in cell number was demonstrated. Cinacalcet HCl affected cell proliferation of CHO cells transfected with CaR, dose dependently, but had no effect on human or mouse osteoblastic cell proliferation in calcium-containing medium (1.6 or 8.6 mM). To test cinacalcet HCl and calcium on osteoclastic cells, peripheral blood mononuclear cells were cultured in medium containing RANK ligand and M-CSF, supplemented with calcium, and/or cinacalcet HCl. Tartrate-resistant acid phosphatase-positive multinucleated osteoclastic cells on plastic or bone were then counted at 11 and 21 days, respectively. Calcium (greater than 6.0 mM) inhibited osteoclast formation, but cinacalcet HCl (30-1000 nM) had no effect on osteoclastic formation or resorption in the presence of calcium (1.6 or 6.1 mM). RT-PCR did not detect CaR in human, rat, or mouse primary osteoblastic cells and cell lines or osteoclastic cells. In conclusion, these studies indicate that the calcium-induced increase in osteoblastic cell number, and the decrease in formation/function of osteoclastic cells, involves a mechanism or receptor other than CaR. In addition, the calcimimetic agent did not potentiate the effects of calcium on normal adult human bone cells in vitro.

Local secretion of parathyroid hormone-related protein by an osteoblastic osteosarcoma (UMR 106-01) cell line results in growth inhibition

Parathyroid hormone-related protein (PTHrP) has been implicated as being important in the growth of tumor cells responsive to the peptide. We utilized a rat osteoblastic osteosarcoma cell line, UMR 106-01, which has PTHrP receptors and a PTHrP-responsive adenylate cyclase/cAMP messenger system, to produce a modified cell line that overexpresses PTHrP. The human PTHrP cDNA sequence was transfected by electroporation into UMR 106-01 cells and the stable cell lines UMR-36 and UMR-34 were established. The modified cell line, UMR-36, had increased levels of PTHrP mRNA compared with control cell lines and secreted PTHrP into the culture medium at levels of 0.01–0.1 pmol/10 7 cells in 12 h. The secreted peptide was biologically active as indicated by its ability to activate adenylate cyclase. The number of UMR-36 cells following 9 days in culture was reduced by up to 80% compared with control lines, which was associated with decreased 3H-thymidine incorporation into genomic DNA. Addition of 1000-fold excess of the PTHrP antagonist, PTHrP(7–34), to UMR-36 cells resulted in the escape of growth inhibition and increased rate of growth. In vivo, tumors derived from UMR-36 cells were smaller in size compared with tumors derived from control cells. In conclusion, increased autocrine secretion of, and responsiveness to, PTHrP results in inhibited growth kinetics of an osteoblast-like bone tumor cell line in vitro and in vivo.

Protein–protein interactions regulate PTH1 receptor signalling

Association of Na+–H+ exchanger regulatory factors (NHERFs) with the parathyroid hormone 1 (PTH1) receptor via a PDZ-domain interaction leads to formation of molecular complexes that enhance Gi/o-mediated phospholipase C (PLC) activation but reduce Gs-mediated signalling.Evidence is accumulating that hormone receptors are present in the cells as part of multimolecular complexes that determine the function of the receptor. Mahon et al. [1xNa+/H+ exchanger regulatory factor 2 directs parathyroid hormone 1 receptor signalling. Mahon, M.J. et al. Nature. 2002; 417: 858–861Crossref | PubMed | Scopus (228)See all References][1] have studied interactions of the C-terminal tail of the PTH1 receptor with other proteins, because earlier data indicated that the C-terminal tail modulates PTH1 receptor signalling. The authors performed yeast two-hybrid screens using the C-terminal tail of the PTH1 receptor as bait. In a human kidney cDNA library, they found that a fragment of Na+–H+ exchanger regulatory factor 2 (NHERF2) interacts strongly with the C-terminal tail of the PTH1 receptor. Because NHERF has been shown to interact with other hormone receptors, and it has two PDZ (PSD95, discs large protein, ZO1) domains that interact with well-defined C-terminal motifs, in addition to an ERM (ezrin, radixin, moesin)-binding motif, the authors have analysed the biological importance of this interaction. In vitro analyses revealed that the C-terminal end of the PTH1 receptor binds to the second PDZ domain (PDZ2) of NHERF2, and a similar interaction is detected with NHERF1. In PS120 Chinese hamster fibroblast cells expressing PTH1 receptor and NHERF2, coprecipitation of these molecules was observed. Most importantly, PTH-induced cAMP stimulation is inhibited when NHERF2 is coexpressed with the PTH1 receptor (from 10–15-fold stimulation without NHERF2 to only 2-fold stimulation with NHERF2), whereas the inositol phosphate (IP) response was markedly increased with NHERF2 coexpression (from 2–4-fold to 20–25-fold). The authors concluded that stimulation of IP production was mediated by Gi/o proteins, because the response was pertussis-toxin sensitive. Because all four isoenzymes of PLCβ, which mediate the hormone-induced generation of IP, contain consensus C-terminal PDZ domain interaction motifs, the authors have used pull-down assays to show that NHERF2 can form multiprotein complexes consisting of PLCβ1 bound to PDZ1 and PTH1 receptor bound to PDZ2. However, it remains to be elucidated whether other subtypes of PLCβ (e.g. PLCβ2) produce similar interactions. Furthermore, the authors have compared the PTH1 receptor-mediated signal generation in two cell lines. They show that in ECV304 cells (a human endothelium-derived cell line with abundant NHERF1 and 2 expression), PTH predominantly increases IP production, whereas in ROS 17/2.8 cells (a rat osteoblastic cell line with no detectable NHERF expression), PTH causes more than a 30-fold increase in cAMP production without a detectable effect on IP signal generation. The appearance of PLC-mediated signalling after transient transfection of NHERF2 into ROS17/2.8 cells was indirectly detected using an AP1-responsive luciferase reporter gene.These data demonstrate that expression of NHERFs leads to formation of molecular complexes that regulate the signalling of the PTH1 receptor. This might be particularly important in polarized cells, because the basal and luminal membranes often have different NHERF expression, and suggests that assembly of receptor complexes with NHERF and possibly other PDZ-domain-containing proteins could be a general mechanism for site-specific regulation of secondary messenger generation.

Effects of Vitamin K2 on Bone of Ovariectomized Rats and on a Rat Osteoblastic Cell Line

The effects of vitamin K₂ on bone mineral density (BMD) and bone metabolic markers of ovariectomized rats, and those on mRNA expression of osteocalcin and IL-6 on a rat osteoblastic cell line, were investigated. BMD and bone metabolic markers were examined in ovariectomized rats after 2 months’ treatment with vitamin K₂, and mRNA expression of osteocalcin and IL-6 were measured in the cell line after 24-hour treatment with vitamin K₂. Vitamin K₂ attenuated the decline in BMD after ovariectomy in the rats, and suppressed serum deoxypyridinoline levels of the ovariectomized rats. No effect on osteocalcin and IL-6 mRNA expression on the cell line was observed. In conclusion, vitamin K₂ has a bone-protective effect on ovariectomized rats.

PTH induction of transcriptional activity of the cAMP response element-binding protein requires the serine 129 site and glycogen synthase kinase-3 activity, but not casein kinase II sites.

We have previously shown that PTH induction of c-fos expression in the rat osteoblastic cell line UMR 106-01 requires the phosphorylation of cAMP response element-binding protein (CREB) at serine 133. Here we show that this event is not sufficient for induced transcriptional activity in UMR cells. Serine 129, but not the casein kinase II sites (serines 108, 111, 114, 117, and 121), also plays a role in the activation of CREB. First, by metabolically labeling an epitope-tagged CREB, we determined that, in addition to serine 133, other residues are phosphorylated in vivo. Using mutational analysis of a GAL4-CREB reporter system we demonstrate that serines 129 and 133 are both required for PTH-induced transcriptional activity, whereas the casein kinase II sites are not. Furthermore, PTH failed to induce transcriptional activity of GAL4-CREB in cells treated with genistein, a general tyrosine kinase inhibitor known to inhibit glycogen synthase kinase-3 (GSK-3) activity, or LiCl, the most specific GSK-3-inhibiting agent known, strongly implicating GSK-3beta in this process. Importantly, although genistein and LiCl each inhibit GSK-3beta activity, neither prevented the phosphorylation of serine 133 induced by PTH. Lastly, when serine 129 is replaced with a negatively charged aspartic acid, LiCl has no effect on the PTH-induced trans-activation of CREB. We propose that GSK-3beta phosphorylates CREB at serine 129 and thus is required for the increased transcriptional activity of CREB in response to PTH.

The cytotoxicity of chromium in osteoblasts: effects on macromolecular synthesis.

Exposure of an immortalized rat osteoblast cell line, FFC cells, to Cr VI resulted in inhibition of protein, DNA and RNA synthesis. Protein synthesis (3H-leucine incorporation) was most sensitive. There was no inhibition of the incorporation of 3H-proline into collagen at the concentrations which inhibited general protein synthesis (1 microM), but synthesis of extracellular collagen fibers was markedly decreased by concentrations of 0.5 microM Cr VI and above. This indicates that some aspect of the post-translational processing of the collagen fibers is sensitive to Cr VI inhibition. Collagen fiber formation was not inhibited by Cr III (which does not penetrate the cell membrane) or when Cr VI was reduced to Cr III extracellularly. This suggests that the Cr VI inhibits an intracellular stage of post-translational collagen processing. Both Cr VI and Cr III inhibit collagenase activity, the former being more potent but less efficacious. Our results suggest that leakage of chromium ions from orthopedic implants may cause a decrease in the proliferation and infiltration of osteoblasts around the implant, and a reduction in the synthesis and altered turnover of collagen in extracellular matrix. These effects will influence the osseointegration of implants, the osteolytic response, and ultimately the stable life-time of the implants.