Cell Line UMR-106 Research Articles

Synthesis of A Tetrahydrocannabinol (THC) Analog For Analyzing Cellular Proliferation in Concert With Epigallocatechin Gallate (EGCG) on A Bone‐like Cancer Cell Line (UMR 106‐01 BSP)

Cannabinoids have been extensively studied in the field of cancer research. Tetrahydrocannabinol (THC) has shown promising results in influencing cellular proliferation when in association with other cannabinoids. This traditional entourage effect solely focuses on the study of THC with other cannabinoids. However, not many studies have been done to explore the synergistic effect of THC analogs when used with non‐cannabinoid compounds. THC in its isolate form for experimentation is very strictly regulated. Therefore, this study was conducted in the pursuit of synthesizing and experimenting with analogs of THC to observe a potential entourage effect with epigallocatechin gallate (EGCG), a compound known for its efficacy to reduce proliferation at higher concentrations in UMR cells. It was hypothesized that active analogs of THC can be synthesized and used in concert with EGCG to potentiate decreased proliferation in the bone‐like cancer cell line UMR 106‐01 BSP (UMR cells). Briefly, a Knoevenagel condensation and a Diels‐Alder reaction using 1,3‐cyclohexanediol dissolved in methanol (MeOH) and citronellal with ethylenediamine diacetic acid (EDDA) at a temperature of 60℃ was used to synthesize a novel THC analog, 3,10,10‐Trimethyl‐1,2,3,4,4a,6,7,8,10,10a‐decahydro‐9‐oxa‐5‐phenanthrenone (TDP). UMR cells were routinely passaged, counted, plated in six‐well culture plates at 480,00 cells/mL, then treated with 10‐fold dilutions of TDP. The plates were incubated for 72 hours in a humidified incubator at 37 degrees Celsius with 5% carbon dioxide infusion. At the end of the experiment, the cells were routinely washed with HANKS buffered saline solution (HBSS), then routinely counted using the Luna Automated Cell Counter. In another experiment, designated cells were co‐treated with TDP+EGCG, following the protocol above. F test ANOVA was used to compare variances and all values in the results were expressed as means ± SD. The results from the attempted cannabinoid analog synthesis yielded a novel active THC analog, TDP. Serial dilutions treatment of the UMR cells with TDP alone showed its ability to decrease cell count in a concentration dependent manner. However, when coupled with higher concentrations of EGCG, the co‐treatment increased cell count rather than potentiating the effect of decreasing cellular proliferation. The F Test ANOVA showed robust statistical significance (p values <0.05) with regard to TDP’s effect of decreasing cell proliferation in UMR cells in a concentration‐dependent manner. Overall, the outcomes of this study suggest that active forms of THC analogs can be synthesized and tested in concert with other non‐cannabinoid compounds like EGCG. This study opens the door to explore the entourage effect of TDP with other non‐cannabinoid compounds that may provide another tool in the therapeutic treatment of bone cancer cells.

Analyses of In-vitro antioxidant and anticancer activity of Cissus quadrangularis stem extract in osteoblastic cell line -UMR- 106

Cancer is one of the leading causes of death worldwide with an estimated 7.8 lakh deaths in 2018 in India. Osteosarcoma, a primary tumour of the bone is the second highest cause of bone cancer related death in young people. It is treated with chemotherapy, radiation, and surgery which cause side effects. Certain traditional plants may have antitumour activities. This research aimed to evaluate the invitro antioxidant and apoptosis induced activity of stem extract of cissus quadrangularis (CQ) on osteoblastic cell line (UMR-106). Various extracts of CQ was subjected to phytochemical screening and antioxidant studies. Ethanolic extract of CQ showed maximum phytoconstituents and better antioxidant activity for both DPPH(2,2-diphenyl-1-picryl-hydrazyl-hydrate)and FRAP(Ferric reducing antioxidant potential) assay. The UMR-106 cell line was treated with different concentrations of the extract ranging from 25μg to 400μg for varying time periods. The dose and time optimization of the ethanolic extract was evaluated in the osteoblastic cell line (UMR-106) using MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) which showed IC50 value with 200μg/ml at 24 hours. There was a reduction in the percentage of cells by 50% in the treated group by SRB assay showing the cytotoxic potency of the extract. Flow cytometry analysis indicated that the extract of CQ induced apoptosis in UMR-106 cell line. Ethanolic extract of CQ has antioxidant effects, also inducing G0/G1 arrest and apoptosis in osteoblasts, suggesting that this might represent a novel and effective agent against bone cancer.

Susceptibility to Localized Corrosion of ASTM F745 and UNS S32750 Stainless Steels Influence of pH and Cytotoxicity Evaluation

Background: Body fluids are highly corrosive as they contain chlorides and hydroxides ions, as well as salts, bacteria, proteins and dissolved oxygen. The pH of the body is usually around 7.4, although this value can vary in a range of 4 to 9 after surgery or because of haematomas, inflammations and infections. ASTM F745 (type 316L) stainless steel has been used for load bearing partial and total joint replacements and post trauma reconstructive surgeries. However, long exposure to the aggressive effect of chloride ion present in the human body, may increase the susceptibility to suffer localized corrosion. Although UNS S32750 has greater corrosion resistance to chloride ion, its magnetic characteristics inhibit its use in implantable devices. Nevertheless, this stainless steel could be used in temporary implants and orthodontic appliances such as brackets, wire arches and bands, due to its high resistance to corrosion, the greater mechanical resistance and the high capacity of plastic forming. Objectives: The objective was to evaluate the susceptibility to localized corrosion in simulated body fluid, in the pH range of 4 to 9. Another objective was to evaluate the cytotoxicity of Cr and Ni present in the chemical composition of both stainless steels. Cytocompatibility was also analysed by seeding cells on the surfaces of both stainless steels. Methods: Cyclic polariation test was performed to evaluate the susceptibility to localized corrosion in 0.9 wt% NaCl aqueous solution, at pH between 4 and 9, maintained at 37°C. For cytotoxicity evaluation, neutral red, MTT and collagen assays were performed using UMR-106 cell line. Cytocompatibility was analysed by seeding UMR-106 cells on the surfaces of both stainless steels. Results: F745-SS was more susceptible to suffer localized corrosion than UNS S32750. Although it showed a tendency to develop transpassive reactions at low pH, galvanostatic tests did not reveal the onset of localized corrosion. The results from the cytotoxicity assays indicated that no adverse effects were observed. UMR-106 osteoblastic cells showed high viability, however, a slight reduction in the collagen production was observed. The cytocompatibility was also satisfactory, since the cells seeded on the surfaces had adequate proliferation. Conclusion: F745-SS is more susceptible to suffer localized corrosion than UNS S32750 in the pH range between 4 and 9. UNS S32750 showed an extensive passive region, however, transpassive reactions were observed at lower pH. On the other hand, no cytotoxic effects were promoted by both stainless steels, although a slight reduction in collagen production was observed. Cells seeded on F745-SS and UNS S32750 surfaces had an acceptable proliferation, without evidence of changes in their morphology.

Synergistic Effects of Erzhi Pill Combined With Methotrexate on Osteoblasts Mediated via the Wnt1/LRP5/β-Catenin Signaling Pathway in Collagen-Induced Arthritis Rats.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by chronic synovitis, bone erosion, and bone loss. Erzhi Pill (EZP), a classic Chinese patent medicine, is often used to treat osteoporosis and shows a capacity for bone metabolism regulation. Methotrexate (MTX), an essential drug for RA treatment, has been reported to inhibit generalized bone loss in RA patients. However, the combined therapeutic effects and mechanism of EZP and MTX in RA have not been fully elucidated. The aim of this study was to investigate the synergistic effect of EZP and MTX on RA and to explore the underlying mechanism through network pharmacological prediction and experimental verification. Chemical compounds of EZP, human target proteins of EZP and MTX, and RA-related human genes were identified in the Encyclopedia of Traditional Chinese Medicine database, PubChem database, and NCBI database, respectively. The molecular network of EZP and MTX in RA was generated and analyzed with Ingenuity Pathway Analysis software according to the datasets. Then, MTX monotherapy, EZP monotherapy, and combined MTX and EZP therapy were administered to collagen-induced arthritis rats, followed by assessment of pathological score, bone damage, bone alkaline phosphatases (BALP), and tartrate-resistant acid phosphatase (TRACP), and of gene levels related to the Wnt1/LRP5/β-catenin pathway according to network pharmacological analysis. Finally, serum samples from MTX-, EZP- and MTX+EZP-treated rats were used to treat the rat osteoblast (OB)-like UMR-106 cell line to evaluate gene levels related to Wnt1/LRP5/β-catenin. Network pharmacological analysis showed that the Wnt/β-catenin signaling pathway was the top signaling pathway shared among MTX, EZP, and RA. The results from in vivo experiments indicated that EZP combined with MTX reduced arthritis severity, alleviated ankle bone damage, increased BALP and decreased TRACP serum levels, and regulated the mRNA expression of Wnt1, LRP5, β-catenin, Runx2, BALP, and BGP in the ankles. In vitro experiments showed that EZP combined with MTX could also improve the expression of genes related to the Wnt1/LRP5/β-catenin pathway. This study demonstrated that EZP in combination with MTX played a synergistic role in regulating OBs in RA, which was connected to the modulatory effect of EZP and MTX on the Wnt1/LRP5/β-catenin signaling pathway.

Sustained Klotho delivery reduces serum phosphate in a model of diabetic nephropathy.

Diabetic nephropathy (DN) is a primary cause of end-stage renal disease and is becoming more prevalent because of the global rise in type 2 diabetes. A model of DN, the db/db uninephrectomized ( db/db-uni) mouse, is characterized by obesity, as well as compromised renal function. This model also manifests defects in mineral metabolism common in DN, including hyperphosphatemia, which leads to severe endocrine disease. The FGF23 coreceptor, α-Klotho, circulates as a soluble, cleaved form (cKL) and may directly influence phosphate handling. Our study sought to test the effects of cKL on mineral metabolism in db/db-uni mice. Mice were placed into either mild or moderate disease groups on the basis of the albumin-to-creatinine ratio (ACR). Body weights of db/db-uni mice were significantly greater across the study compared with lean controls regardless of disease severity. Adeno-associated cKL administration was associated with increased serum Klotho, intact, bioactive FGF23 (iFGF23), and COOH-terminal fragments of FGF23 ( P < 0.05). Blood urea nitrogen was improved after cKL administration, and cKL corrected hyperphosphatemia in the high- and low-ACR db/db-uni groups. Interestingly, 2 wk after cKL delivery, blood glucose levels were significantly reduced in db/db-uni mice with high ACR ( P < 0.05). Interestingly, several genes associated with stabilizing active iFGF23 were also increased in the osteoblastic UMR-106 cell line with cKL treatment. In summary, delivery of cKL to a model of DN normalized blood phosphate levels regardless of disease severity, supporting the concept that targeting cKL-affected pathways could provide future therapeutic avenues in DN. NEW & NOTEWORTHY In this work, systemic and continuous delivery of the "soluble" or "cleaved" form of the FGF23 coreceptor α-Klotho (cKL) via adeno-associated virus to a rodent model of diabetic nephropathy (DN), the db/db uninephrectomized mouse, normalized blood phosphate levels regardless of disease severity. This work supports the concept that targeting cKL-affected pathways could provide future therapeutic avenues for the severe mineral metabolism defects associated with DN.

Epigenetic Regulation of BMP2 by 1,25-dihydroxyvitamin D3 through DNA Methylation and Histone Modification

Genetic hypercalciuric stone-forming (GHS) rats have increased intestinal Ca absorption, decreased renal tubule Ca reabsorption and low bone mass, all of which are mediated at least in part by elevated tissue levels of the vitamin D receptor (VDR). Both 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and bone morphogenetic protein 2 (BMP2) are critical for normal maintenance of bone metabolism and bone formation, respectively. The complex nature of bone cell regulation suggests a potential interaction of these two important regulators in GHS rats. In the present study, BMP2 expression is suppressed by the VDR-1,25(OH)2D3 complex in Bone Marrow Stromal Cells (BMSCs) from GHS and SD rat and in UMR-106 cell line. We used chromatin immunoprecipitation (ChIP) assays to identify VDR binding to only one of several potential binding sites within the BMP2 promoter regions. This negative region also mediates suppressor reporter gene activity. The molecular mechanisms underlying the down-regulation of BMP2 by 1,25(OH)2D3 were studied in vitro in BMSCs and UMR-106 cells using the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) and the histone deacetylase inhibitor trichostatin A (TSA). Both DAC and TSA activate BMP2 expression in combination with 1,25(OH)2D3. Bisulfite DNA pyrosequencing reveals 1,25(OH)2D3 to completely hypermethylate a single CpG site in the same BMP2 promoter region identified by the ChIP and reporter gene assays. ChIP assays also show that 1,25(OH)2D3 can increase the repressive histone mark H3K9me2 and reduce the acetylation of histone H3 at the same BMP2 promoter region. Taken together, our results indicate that 1,25(OH)2D3 binding to VDR down-regulates BMP2 gene expression in BMSCs and osteoblast-like UMR-106 cells by binding to the BMP2 promoter region. The mechanism of this 1,25(OH)2D3-induced transcriptional repression of BMP2 involves DNA methylation and histone modification. The study provides novel evidence that 1,25(OH)2D3 represses bone formation through down-regulating BMP2 expression both in vivo and in vitro.

Loading-related Regulation of Transcription Factor EGR2/Krox-20 in Bone Cells Is ERK1/2 Protein-mediated and Prostaglandin, Wnt Signaling Pathway-, and Insulin-like Growth Factor-I Axis-dependent

Of the 1,328 genes revealed by microarray to be differentially regulated by disuse, or at 8 h following a single short period of osteogenic loading of the mouse tibia, analysis by predicting associated transcription factors from annotated affinities revealed the transcription factor EGR2/Krox-20 as being more closely associated with more pathways and functions than any other. Real time quantitative PCR confirmed up-regulation of Egr2 mRNA expression by loading of the tibia in vivo. In vitro studies where strain was applied to primary cultures of mouse tibia-derived osteoblastic cells and the osteoblast UMR106 cell line also showed up-regulation of Egr2 mRNA expression. In UMR106 cells, inhibition of β1/β3 integrin function had no effect on strain-related Egr2 expression, but it was inhibited by a COX2-selective antagonist and imitated by exogenous prostaglandin E2 (PGE2). This response to PGE2 was mediated chiefly through the EP1 receptor and involved stimulation of PKC and attenuation by cAMP/PKA. Neither activators nor inhibitors of nitric oxide, estrogen signaling, or LiCl had any effect on Egr2 mRNA expression, but it was increased by both insulin-like growth factor-1 and high, but not low, dose parathyroid hormone and exogenous Wnt-3a. The increases by strain, PGE2, Wnt-3a, and phorbol 12-myristate 13-acetate were attenuated by inhibition of MEK-1. EGR2 appears to be involved in many of the signaling pathways that constitute early responses of bone cells to strain. These pathways all have multiple functions. Converting their strain-related responses into coherent “instructions” for adaptive (re)modeling is likely to depend upon their contextual activation, suppression, and interaction probably on more than one occasion.

Biological effects of magnesium particles degradation on UMR-106 cell line: Influence of fluoride treatments

Mg-based materials are promising for orthopedic, dental, and cardiovascular applications but their high degradation rate in vivo (release of Mg ions and debris particles) is cause of great concern. Protective treatments involving fluoride conversion coatings have been proposed in order to reduce corrosion rates. The aim of this study was to evaluate Mg debris biodegradation and its possible cytotoxic effects on osteoblastic cells in situ. Neutral Red dying and Acridine Orange staining techniques were used as endpoints to analyse the cytotoxic effects at 100–2000 μg/mL concentration range. Results showed a marked variation of Mg ion concentration in the culture medium after different exposure periods (1, 2, or 24 h). Interestingly, the release rate of magnesium ions was dependent on the presence or absence fluoride treatment. Adverse effects induced by ≥1000 μg/mL MP doses and Mg ion concentrations higher than 480 μg/mL were observed on cells. Results showed significant differences between the concentration of Mg ions in the presence and absence of cells. This fact reveals a dynamic equilibrium mediated by Mg ion input and output in the cells that leads to the change in MP corrosion rates. Fluoride release from conversion coatings did not show cytotoxic effects.

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.

Potential antitumor properties of a protein isolate obtained from the seeds of Amaranthus mantegazzianus

Amaranth is a crop that can be grown in different soils and climates, being resistant to high temperatures, drought, and some pests. The amaranth plant has nutritional qualities and desirable biological properties. The aim of the study is to investigate the potential antitumor properties of Amaranthus-mantegazzianus-protein isolate (MPI) and to elucidate the possible mechanism of action. We use four different tumor-derived and in vitro-transformed cell lines with different morphology and tumorigenicity (MC3T3E1, UMR106, Caco-2, and TC7). The MPI showed an antiproliferative effect on four cell lines with different potencies. The tumor-cell line UMR106 was the most sensitive (IC(50): 1 mg/ml). This antiproliferative effect of the MPI was enhanced by protease treatment (IC(50): 0.5 after 30% hydrolysis). In addition, the MPI produced morphological changes and caused a rearrangement of the cytoskeleton in UMR106 cell line. In an attempt to elucidate the mechanism of action, we observed that the MPI inhibited cell adhesion and induced apoptosis and necrosis in the UMR106 cell line. In reversibility studies, we were able to observe both temporary and permanent cytostatic and cytotoxic effects on the part of the MPI, depending on its concentration. we report a protein isolate from the seeds of Amaranthus mantegazzianus that exhibit potential antitumor properties and propose a putative mechanism of action.

Establishment of reproducible osteosarcoma rat model using orthotopic implantation technique

In experimental musculoskeletal oncology, there remains a need for animal models that can be used to assess the efficacy of new and innovative treatment methodologies for bone tumors. Rat plays a very important role in the bone field especially in the evaluation of metabolic bone diseases. The objective of this study was to develop a rat osteosarcoma model for evaluation of new surgical and molecular methods of treatment for extremity sarcoma. One hundred male SD rats weighing 125.45+/-8.19 g were divided into 5 groups and anesthetized intraperitoneally with 10% chloral hydrate. Orthotopic implantation models of rat osteosarcoma were performed by injecting directly into the SD rat femur with a needle for inoculation with SD tumor cells. In the first step of the experiment, 2x10(5) to 1x10(6) UMR106 cells in 50 microl were injected intraosseously into median or distal part of the femoral shaft and the tumor take rate was determined. The second stage consisted of determining tumor volume, correlating findings from ultrasound with findings from necropsia and determining time of survival. In the third stage, the orthotopically implanted tumors and lung nodules were resected entirely, sectioned, and then counter stained with hematoxylin and eosin for histopathologic evaluation. The tumor take rate was 100% for implants with 8x10(5) tumor cells or more, which was much less than the amount required for subcutaneous implantation, with a high lung metastasis rate of 93.0%. Ultrasound and necropsia findings matched closely (r=0.942; p<0.01), which demonstrated that Doppler ultrasonography is a convenient and reliable technique for measuring cancer at any stage. Tumor growth curve showed that orthotopically implanted tumors expanded vigorously with time-lapse, especially in the first 3 weeks. The median time of survival was 38 days and surgical mortality was 0%. The UMR106 cell line has strong carcinogenic capability and high lung metastasis frequency. The present rat osteosarcoma model was shown to be feasible: the take rate was high, surgical mortality was negligible and the procedure was simple to perform and easily reproduced. It may be a useful tool in the investigation of antiangiogenic and anticancer therapeutics. Ultrasound was found to be a highly accurate tool for tumor diagnosis, localization and measurement and may be recommended for monitoring tumor growth in this model.

Parathyroid hormone regulates osterix and Runx2 mRNA expression predominantly through protein kinase A signaling in osteoblast-like cells

Runt-related transcription factor 2 (Runx2) and osterix are osteoblast-specific transcription factors essential for the development of osteoblastic cells and bone formation. PTH given intermittently has anabolic effects on bone; however, the exact role remains to be understood completely. The purpose of this study was both to investigate whether PTH regulates Runx2 as well as osterix expression and to identify the signaling used. Using RT-PCR, we confirmed that PTH (1-34) regulated Runx2 and osterix mRNA expression, in rat osteoblast-like cell line UMR 106, in a dose- and time-dependent manner. PTH in low concentrations stimulated both Runx2 and osterix mRNA expression while that in high concentrations did not. Forskolin, an adenylate cyclase activator, also enhanced Runx2 and osterix transcription, and the stimulatory effects of PTH and forskolin were blocked by the pre-treatment of the cells with H-89, a protein kinase A (PKA) inhibitor. In contrast, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) had no effect on Runx2 transcription, but induced an increase in osterix mRNA level at the concentration of 500 nM at 12 h after treatment. Moreover, pre-treatment of the cells with calphostin C, a PKC-specific inhibitor, reduced the increase in osterix transcripts enhanced by PTH and PMA 12 h after treatment. However, these inhibitory effects were not sustained for longer terms. These observations demonstrate that PTH stimulates Runx2 and osterix expression in vitro, at least in part, at transcriptional level. Induction of Runx2 mRNA is mediated through the activation of cAMP/PKA signal transduction. In the case of osterix, although the increase in mRNA level is predominantly mediated via cAMP/PKA signaling, PKC activation might also be involved in this process.

HMGB1 expression and release by bone cells

Immune and bone cells are functionally coupled by pro-inflammatory cytokine intercellular signaling networks common to both tissues and their crosstalk may contribute to the etiologies of some immune-associated bone pathologies. For example, the receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK) signaling axis plays a critical role in dendritic cell (DC) function as well as bone remodeling. The expression of RANKL by immune cells may contribute to bone loss in periodontitis, arthritis, and multiple myeloma. A recent discovery reveals that DCs release the chromatin protein high mobility group box 1 (HMGB1) as a potent immunomodulatory cytokine mediating the interaction between DCs and T-cells, via HMGB1 binding to the membrane receptor for advanced glycation end products (RAGE). To determine whether osteoblasts or osteoclasts express and/or release HMGB1 into the bone microenvironment, we analyzed tissue, cells, and culture media for the presence of this molecule. Our immunohistochemical and immunocytochemical analyses demonstrate HMGB1 expression in primary osteoblasts and osteoclasts and that both cells express RAGE. HMGB1 is recoverable in the media of primary osteoblast cultures and cultures of isolated osteoclast precursors and osteoclasts. Parathyroid hormone (PTH), a regulator of bone remodeling, attenuates HMGB1 release in cultures of primary osteoblasts and MC3T3-E1 osteoblast-like cells but augments this release in the rat osteosarcoma cell line UMR 106-01, both responses primarily via activation of adenylyl cyclase. PTH-induced HMGB1 discharge by UMR cells exhibits similar release kinetics as reported for activated macrophages. These data confirm the presence of the HMGB1/RAGE signaling axis in bone.

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.

Two new pregnane glycosides from Dioscorea futschauensis R. KUNTH.

Two new pregnane glycosides (1, 2) together with two known saponins were isolated from the rhizomes of Dioscorea futschauensis R. KUNTH. The structures of 1 and 2 were established as 16alpha-methoxyl-3beta-[(O-alpha-L-rhamnopyranosyl-(1-->2)-O-[alpha-L-rhamnopyranosyl-(1-->4)]-beta-D-glucopyranosyl)oxy]pregn-5-en-20-one and 21-methoxyl-3beta-[(O-alpha-L-rhamnopyranosyl-(1-->2)-O-[alpha-L-rhamnopyranosyl-(1-->4)]-beta-D-glucopyranosyl)oxy]pregn-5,16-en-20-one, respectively, on the basis of two-dimension NMR (2D NMR) and other spectral analysis. Their in vitro bioactivity against plant pathogenic fungus Pyricularia oryzae and osteoblastic proliferation stimulatory activity in the UMR106 cell line were evaluated.

Osteoblastic Proliferative Activity of Extracts of Qing’e pill and its Disassembled Formulae

Qing’e pill is one of the famous traditional Chinese compound prescriptions used to treat bone diseases. It contains four botanical drugs, i.e., the cortex of Eucommia ulmoides Oliv., the fruit of Psoralea corylifolia L., the seed of Juglans regia L. and the rhizome of Allium sativum L. In this study, osteoblastic proliferation activity of extracts from Qing’e pill and its disassembled formulae was investigated with the osteoblast-like UMR106 cell line as a model. The extract of Qing’e pill, both alcohol and aqueous, stimulated cell proliferation in a dose-responsive manner. The proliferative activity of alcohol extract was more potent than that of the aqueous one with a maximal growth stimulation ratio (GSR) of 58.5% versus 38.8%. Eucommia ulmoides and Psoralea corylifolia produced a maximum proliferative promotion of 38.7% and 34.0%, respectively, when co-cultured with UMR106 cells. Neither Juglans regia nor Allium sativum showed a significant effect on osteoblastic proliferation. When Eucommia ulmoides and Psoralea corylifolia were combined, the stimulating action (GSR = 47.2%) became stronger than that of either individual drug. The enhancement effect was more marked when Juglans regia or both Juglans regia and Allium sativum were added. These results demonstrated the synergy of the four botanical drugs and the rationality of Qing’e pill prescription in a modern scientific way. This is the first time to study the stimulating osteroblastic proliferation effect of a traditional Chinese compound prescription on the basis of disassembled formulae.

Characterization of an Osteoblast-specific Enhancer Element in the CBFA1 Gene

Cbfa1 is a critical regulator of cell differentiation expressed only in the osteochondrogenic lineage. To define the molecular basis of this cell-specific expression we analyzed the murine Cbfa1 promoter. Here we show that the first 976 bp of this promoter are specifically active in osteoblastic cells. Within this region DNase I footprinting delineated a 40-bp area (CE1) protected differently by nuclear extracts from osteoblastic cells and from non-osteoblastic cells. When multimerized, CE1 conferred an osteoblast-specific activity to a heterologous promoter in DNA transfection experiments; this enhancing ability was conserved between mouse, rat, and human CE1 present in the respective Cbfa1 promoters. CE1 site-specific mutagenesis determined that it binds NF1- and AP1-like activities. Further analyses revealed that the NF1 site acts as a repressor in non-osteoblastic cells due to the binding of NF1-A, a NF1 isoform not expressed in osteoblastic cells. In contrast, the AP1 site mediates an osteoblast-specific activation caused by the preferential binding of FosB to CE1 in osteoblastic cells. In summary, this study identified an osteoblast-specific enhancer in the Cbfa1 promoter whose activity is achieved by the combination of an inhibitory and an activatory mechanism.

Overexpression of BMP-2 Modulates Morphology, Growth, and Gene Expression in Osteoblastic Cells

Bone morphogenetic proteins (BMP) play a pivotal role in growth and differentiation of osteoblastic lineage cells. BMPs are potent stimulators of bone formation in various animal models. To understand the mechanism of BMP action in bone cells, we have investigated the effects of overexpression of the BMP-2 gene on proliferation and differentiation of UMR-106 rat osteosarcoma cells. A stable UMR-106 cell line overexpressing the BMP-2 gene was established by transfection of cells using a mammalian expression vector harboring human BMP-2 cDNA followed by G418 selection. After introduction of the BMP-2 gene, UMR-106 cells appeared more spindle-shaped in morphology compared to the predominantly cuboidal appearance of the parental cells. Overexpression of BMP-2 markedly inhibited proliferation as measured by cell counting and [3H]thymidine incorporation assays. Extracellular matrix (ECM) derived from cells overexpressing BMP-2 exhibited a less supportive effect on proliferation of UMR cells than did ECM derived from parental cells. Furthermore, cell–cell communication through gap junctions was reduced more than 50% as determined by nondisruptive fluorescent dye transfer assays. Overexpression of BMP-2 significantly stimulated expression of osteocalcin and alkaline phosphatase genes, indicating its role in osteoblastic differentiation. There was little effect on osteopontin gene expression.

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.

Mechanisms of regulation of G(11)alpha protein by dexamethasone in osteoblastic UMR 106-01 cells.

We have previously demonstrated that glucocorticoids increased G(q/11)alpha protein expression and phospholipase C activity in the rat osteosarcoma cell line UMR 106-01. In this study, we demonstrated that G(11)alpha is the primary G(q)-subtype family member expressed in UMR cells. Dexamethasone treatment increased the expression of G(11)alpha protein in both a time- and a dose-dependent manner. Glucocorticoid treatment significantly increased the half-life of G(11)alpha protein from 20.3 to 63 h. Steady-state G(11)alpha mRNA level was also increased by glucocorticoid treatment by approximately 70%. This change was not the result of changes in RNA stability but rather the result of increased transcription, because the glucocorticoid-mediated upregulation of G(11)alpha mRNA was blocked by the transcription inhibitor actinomycin D. The dexamethasone induction of G(11)alpha mRNA occurred after a time lag of 12-24 h and was blocked by the protein synthesis inhibitor cycloheximide. These results suggest that the dexamethasone-induced rise in G(11)alpha protein results primarily from changes in the degradation rate of the protein, whereas changes in G(11)alpha mRNA play a smaller role and require de novo synthesis of regulatory protein(s).