OBJECTIVETo study the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) mRNA in mouse osteoblasts.

METHODSCalvariae derived from CD-1 neonatal mouse (after born 24 h). Bone samples were processed by the collagenase/trypsin digestion method. Mouse osteoblasts were cultured in vitro. After 48 hours of addition of 1,25(OH)2D3 (0, 10(-8), 10(-9), 10(-11) mol/L) to the culture medium of mouse osteoblasts, the content of the OPG protein in culture medium was estimated with enzyme linked immunosorbent assay. Total RNA was prepared from mouse osteoblasts. mRNA expression of OPG and RANKL were detected by reverse transcription-polymerase chain reaction.

RESULTSThe mRNA expression of OPG in osteoblasts added with 1,25(OH)2D3 significantly decreased compared with the controls, which was markedly dose-dependent. OPG protein production in the medium decreased after treatment with 1,25(OH)2D3. In contrast, RANKL mRNA expression levels in osteoblasts significantly increased after 48 h of culture with 1,25(OH)2D3.

CONCLUSION1,25 (OH)2D3 can stimulate RANKL mRNA expression, but decrease OPG mRNA levels in vitro in mouse osteoblasts.

Animals ; Animals, Newborn ; Calcitriol ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; Glycoproteins ; biosynthesis ; genetics ; physiology ; Ligands ; Membrane Glycoproteins ; biosynthesis ; genetics ; Mice ; NF-kappa B ; biosynthesis ; genetics ; Osteoclasts ; metabolism ; physiology ; Osteoprotegerin ; RANK Ligand ; RNA, Messenger ; biosynthesis ; genetics ; Receptor Activator of Nuclear Factor-kappa B ; Receptors, Cytoplasmic and Nuclear ; analysis ; biosynthesis ; genetics ; physiology ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

OBJECTIVETo investigate the effects of LPS and/or TNF-alpha on periodontal ligament cell (PDLC) proliferation and OPG secretion.

METHODSHealthy premolars extracted for orthodontic reasons from a 12 years old boy were obtained, and periodontal tissues were collected and cultured to obtain PDLCs. Cloned PDLCs were obtained by means of limited dilutions, and were characterized as follows: alkaline phosphatase activity, collagen III production and bone-like nodules formation. LPS and rhTNF-alpha were added into culture media and their effects on PDLC proliferation and OPG secretion were observed. The OPG concentrations in cell culture supernatants were detected by sandwich ELISA. Living cell numbers were demonstrated by MTT test. The average levels of OPG secretion by a single cell were calculated by dividing OPG concentration with MTT result.

RESULTSrhTNF-alpha above 10 micro g/L decreased the mtt and opg detecting results, but increased the opg/mtt values (P < 0.05). However, LPS had no effect on mtt, opg or opg/mtt values. Neither it had any interaction with rhTNF-alpha (P > 0.05).

CONCLUSIONSTNF-alpha prohibits the proliferation of PDLCs but enhances their OPG secretion. However, LPS has no effect on neither side. Our works support the hypothesis that there may be an inverse feedback regulation pattern of increasing periodontal OPG production against local bone resorption activity. PDLCs might not be the natural target cells of LPS' direct cytotoxic effect.

Child ; Glycoproteins ; biosynthesis ; drug effects ; Humans ; In Vitro Techniques ; Lipopolysaccharides ; pharmacology ; Male ; Osteoprotegerin ; Periodontal Ligament ; metabolism ; pathology ; Receptors, Cytoplasmic and Nuclear ; biosynthesis ; drug effects ; Receptors, Tumor Necrosis Factor ; biosynthesis ; drug effects ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVEThis study was carried out to investigate the effects of insulin-like growth factor-II (IGF-II) and basic fibroblast growth factor (bFGF) on osteoprotegerin (OPG) secretion of periodontal ligament cells (PDLCs).

METHODSHealthy human premolars extracted for orthodontic reasons from 12-14 years old donators were obtained, and periodontal tissues were collected and cultured to obtain PDL cells. Primary or first passage PDLCs were cloned by means of limited dilutions. PDLCs with osteoblastic phenotypes were characterized as follows: Alkaline phosphatase activity, collagen III production and bone-like nodules formation. IGF-II and bFGF were added into culture media and their effects on PDLCs proliferation and OPG secretion were observed. The OPG concentrations in cell culture supernatants were detected by sandwich ELISA. Living cell numbers were demonstrated by MTT test. The average levels of OPG secretion by a single cell were calculated by dividing OPG concentration with MTT-test result.

RESULTSBoth IGF-II and bFGF upregulated the mtt values (P < 0.05), but ICF-II downregulated the opg/mtt values (P < 0.05), whereas bFGF had no significant effect on opg/mtt values (P > 0.05).

CONCLUSIONIGF-II enhances the proliferation of PDL cells but prohibits OPG secretion. Although bFGF has the same effect on the proliferation of PDL cells, it has no effect on OPG secretion. Before cytokines were used to enhance periodontal regeneration, their effects on local bone balance should also be studied.

Adolescent ; Cells, Cultured ; Child ; Fibroblast Growth Factor 2 ; pharmacology ; Glycoproteins ; biosynthesis ; Humans ; Insulin-Like Growth Factor II ; pharmacology ; Osteoprotegerin ; Periodontal Ligament ; cytology ; drug effects ; metabolism ; Receptors, Cytoplasmic and Nuclear ; biosynthesis ; Receptors, Tumor Necrosis Factor ; biosynthesis

OBJECTIVETo express human osteoprotegerin (OPG) in E. Coli and analyze its bioactivity in vitro.

METHODSSynthetic oligonucleotides were used to amplify human OPG gene by RT-PCR from total RNA of human osteosarcoma cell line MG63. The OPG cDNA coding for 380 amino acid residues was inserted into prokaryotic expression vector pRSET-A, transformed into competent E. Coli BL21, and induced by 0.1 mmol/l IPTG. SDS-PAGE and Western blot were performed to identify OPG-6His fusion protein. After purified by affinity chromatography, 1,000 microg/L or 1,500 microg/L of OPG-6His were added into the mouse bone marrow cells culture medium. The number of tartrate-resistant acid phophatase (TRAP)-positive multinucleated cells and resorption pits were counted to assess the bioactivity of expression products.

RESULTSThe sequence of OPG mature peptide encoding cDNA obtained in this experiment was as same as reported. SDS-PAGE showed 24% of total bacterial protein was of OPG-6His fusion protein. Western blot assay demonstrated that the molecular weight of recombinant protein was about 46 KD and could react specifically with human anti-OPG antibody. The mouse bone marrow cells were induced by 1alpha, 25-dihydroxyvitaminD3 (10(-8) mol/L) and Dexamethasone (10(-7) mol/L) to form osteoclastic-like multinucleated cells. 1,500 microg/L of purified OPG-6His protein could decrease the number of resorption pits and TRAP-positive multinucleated cells in vitro (P < 0.05), but it didn't show the same effects when the concentration of OPG-6His fusion protein was of 1,000 microg/L.

CONCLUSIONSHuman OPG-6His fusion protein is expressed and purified in E. Coli. The expression products have moderate inhibitory effects on osteoclast differentiation and bone resorption in vitro only when excessive amount of proteins are added into the culture medium, indicating that prokaryotic expression of fuctionalal OPG protein awaits further investigation.

Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cloning, Molecular ; Escherichia coli ; genetics ; Glycoproteins ; biosynthesis ; genetics ; Humans ; Osteoclasts ; drug effects ; physiology ; Osteoprotegerin ; Receptors, Cytoplasmic and Nuclear ; biosynthesis ; genetics ; Receptors, Tumor Necrosis Factor ; Recombinant Fusion Proteins ; biosynthesis ; pharmacology

OBJECTIVETo explore the effect of Herba Epimedii flavone (HEF) on the osteoblast metabolism in vitro.

METHODOsteoblast were obtained from new born rat calvaria by digestive enzymes. MTF, PNPP and RT-PCR were used to observe the proliferation, activity of ALP and mRNA expression of OPG and RANKL of cultured osteoblasts in vitro.

RESULTIt was found that HEF had the effect on stimulating cell proliferation, activity of ALP and the mRNA expression of OPG of cultured osteoblasts (P < 0.01, P < 0.05).

CONCLUSIONHEF can promote the proliferation, the differentiation and the expression of OPG mRNA of the osteoblasts cultured in vitro.

Alkaline Phosphatase ; metabolism ; Animals ; Animals, Newborn ; Carrier Proteins ; biosynthesis ; genetics ; Cell Proliferation ; drug effects ; Cells, Cultured ; Epimedium ; chemistry ; Flavones ; isolation & purification ; pharmacology ; Glycoproteins ; biosynthesis ; genetics ; Membrane Glycoproteins ; biosynthesis ; genetics ; Osteoblasts ; cytology ; metabolism ; Osteoprotegerin ; Plants, Medicinal ; chemistry ; RANK Ligand ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, Cytoplasmic and Nuclear ; biosynthesis ; genetics ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics

This study is to evaluate the cytotoxicity of mitomycin C (MMC) and its analogue 5-(aziridin-1-yl)-3-hydroxymethyl-1-methylindole-4,7-dione (629) as well as the effect of transfection of constitutive androstane receptor (CAR) on their biological effects. HepG2 cells were transfected with the plasmids mCAR1/pCR3 mediated by liposome. Vector pCR3 was used as control. Transfected cells were screened by G418 resistance and limiting dilution. The expressions of plasmid mCAR1/pCR3 and CYP2B6 mRNA were detected by RT-PCR; Cytotoxicities of MMC and 629 in vitro were evaluated in g2car cells and HepG2 cells by MTT method under anaerobic and aerobic conditions. mRNA expression of CAR and CYP2B6 can not be detected in HepG2 cells and HepG2/pCR3 cells but can in g2car cells. It is shown that plasmid mCAR1/pCR3 was transfected into g2car cells successfully and target CYP2B6 was transactivated by CAR. To compare with aerobic and anaerobic, the cytotoxicities of MMC and 629 to HepG2 cells and g2car cells had significantly enhanced (P < 0.05), and transfect CAR gene can improve the cytotoxicity of MMC (P < 0.05), but not 629 (P > 0.05). Furthermore, CYP2B6 is one master enzyme for the metabolism of MMC and not 629. Transfection of CAR can increase expression of CYP2B6 mRNA in HepG2 cells, and can affect cytotoxicities of MMC and 629.
Antibiotics, Antineoplastic ; pharmacology ; Aryl Hydrocarbon Hydroxylases ; biosynthesis ; genetics ; Aziridines ; pharmacology ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Death ; drug effects ; Cell Hypoxia ; Cell Line, Tumor ; Cytochrome P-450 CYP2B6 ; Humans ; Indoles ; pharmacology ; Inhibitory Concentration 50 ; Liver Neoplasms ; metabolism ; pathology ; Mitomycin ; pharmacology ; Oxidoreductases, N-Demethylating ; biosynthesis ; genetics ; Plasmids ; RNA, Messenger ; metabolism ; Receptors, Cytoplasmic and Nuclear ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics ; Transfection

Bone destruction is primarily mediated by osteoclastic bone resorption, and cancer cells stimulate the formation and activation of osteoclasts next to metastatic foci. Accumulating evidences indicate that receptor activator of NF-kB ligand (RANKL) is the ultimate extracellular mediator that stimulates osteoclast differentiation into mature osteoclasts. In contrast, osteoprotegerin (OPG) inhibits osteoclast development. In order to elucidate a mechanism for cancer-induced osteoclastogenesis, cells from a human breast cancer line, MDA-MB-231, were directly co-cultured with ST2, MC3T3-E1, or with primary mouse calvarial cells. Osteoclast-like cells and tartarate resistant acid phosphatase (TRAP) activities were then quantitated. We examined these cell lines and samples from breast cancer by RT-PCR for the expressions of OPG and RANKL mRNA. Compared to controls, co-culture of MDA-MB-231 cells with stromal or osteoblastic cells induced an increase in number of osteoclasts and TRAP activities. MDA-MB-231 cells alone or breast cancer samples did not express RANKL mRNA. However, co-culture of these cancer cells with stromal or osteoblastic cells induced RANKL mRNA expression and decreased OPG mRNA expression. These experiments demonstrate that direct interactions between breast cancer and stromal or osteoblastic cells induce osteoclastogenesis in vitro through modulating RANKL expression.
3T3 Cells ; Acid Phosphatase/metabolism ; Animals ; Bone Neoplasms/*metabolism/*secondary ; Breast Neoplasms/*pathology ; Carrier Proteins/*biosynthesis ; Cell Differentiation ; Cell Line, Tumor ; Cells, Cultured ; Coculture ; Culture Media, Conditioned/pharmacology ; Glycoproteins/*biosynthesis ; Human ; Isoenzymes/metabolism ; Male ; Membrane Glycoproteins/*biosynthesis ; Mice ; Mice, Inbred C57BL ; Neoplasm Metastasis ; Osteoblasts/metabolism ; Osteoclasts/metabolism ; Protein Binding ; RNA, Messenger/metabolism ; Receptors, Cytoplasmic and Nuclear/*biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

High ambient Ca2+ at bone resorption sites have been implicated to play an important role in the regulation of bone remodeling. The present study was performed to clarify the mode of high extracellular Ca2+ (Ca2+e)-induced modulation of osteoclastogenesis and the expression of receptor activator of nuclear factor-kB ligand (RANKL) and osteoprotegerin (OPG), thereby to define its role in osteoclast formation. Mouse bone marrow cells were cocultured with osteoblastic cells in the absence or presence of osteoclastogenic factors such as 1,25-dihydroxyvitaminD3 (1,25-(OH)2vitD3) and macrophage colony-stimulating factor/soluble RANKL. Ca2+ concentration in media (1.8 mM) was adjusted to 3, 5, 7 or 10 mM. Osteoclast formation was confirmed by the appearance of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells and the expression of osteoclast phenotypic markers (calcitonin receptor, vitronectin receptor, cathepsin K, matrix metalloproteinase-9, carbonic anhydrase 2). High Ca2+e alone significantly stimulated osteoclast formation in a dose-dependent manner. However, in the presence of highly osteoclastogenic factors, high Ca2+e significantly inhibited osteoclastogenesis. High Ca2+e alone continuously up-regulated RANKL expression while only transiently increased OPG expression. However, in the presence of 1,25-(OH)2vitD3, high Ca2+e did not change the 1,25-(OH)2vitD3- induced RANKL expression while increased OPG expression. Taken together, these findings suggest that high Ca2+e alone increase osteoclastogenesis but inhibit in the presence of other osteoclastogenic factors. In addition, high Ca2+e-induced osteoclastogenesis may be mediated by osteoblasts via up-regulation of RANKL expression. Meanwhile up-regulated OPG might participate in the inhibitory effect of high Ca2+e on 1,25-(OH)2vitD3-induced osteoclastogenesis.
Animals ; Bone Marrow Cells/metabolism/physiology ; Bone Remodeling ; Calcium/*metabolism ; Carrier Proteins/biosynthesis ; Cations, Divalent ; Cells, Cultured ; Coculture ; Extracellular Space/*metabolism ; Glycoproteins/biosynthesis ; Membrane Glycoproteins/biosynthesis ; Mice ; Mice, Inbred ICR ; Osteoblasts/*cytology/metabolism ; Osteoclasts/*cytology/metabolism ; Receptors, Cytoplasmic and Nuclear/biosynthesis ; Vitamin D/*analogs & derivatives/pharmacology

OBJECTIVETo study the expression of type I inositol 1,4,5-triphosphate receptor in rat glomerular and afferent arterioles in a model of liver cirrhosis and study the role of cross-membrane message transduction in the pathogenesis of hepatorenal syndrome.

METHODSIn a rat model of carbontetrachloride liver cirrhosis, the expression of type I inositol 1,4,5-triphosphate receptor (IP3R) on glomerular and afferent arterioles was measured by immunohistochemical method.

RESULTSIn the experimental group, 30 rats were used to make a model of liver cirrhosis. 11 rats survived during the experiment. The expression of type I IP3R on glomerular and afferent arterioles was 4.97+/-1.34 and 4.09+/-1.14 in the liver cirrhosis group, and it was 2.43+/-1.67 and 1.83+/-1.32 in the normal control rats. The differences between these two groups are statistically significant (t = 2.28, P = 0.0458).

CONCLUSIONExpression of type I IP3 receptor on rat glomerular and afferent arterioles in a model of liver cirrhosis indicated that the mechanism of cross-membrane message transduction plays a very important role in the pathogenesis of hepatorenal syndrome.

Animals ; Arterioles ; metabolism ; Calcium Channels ; biosynthesis ; genetics ; Carbon Tetrachloride ; Carbon Tetrachloride Poisoning ; Inositol 1,4,5-Trisphosphate Receptors ; Kidney ; metabolism ; Kidney Glomerulus ; blood supply ; metabolism ; Liver Cirrhosis, Experimental ; chemically induced ; complications ; metabolism ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Receptors, Cytoplasmic and Nuclear ; biosynthesis ; genetics ; Renal Artery ; metabolism

OBJECTIVE[corrected] To investigate the effect of peroxisome proliferator-activated receptors (PPARs) activators on plasminogen activator inhibitor type-1 (PAI-1) expression in human umbilical vein endothelial cells and the possible mechanism.

METHODSHuman umbilical vein endothelial cells (HUVECs) were obtained from normal fetus, and cultured conventionally. Then the HUVECs were exposed to test agents (linolenic acid, linoleic acid, oleic acid, stearic acid and prostaglandin J2 respectively) in varying concentrations with fresh media. RT-PCR and ELISA were applied to determine the expression of PPARs and PAI-1 in HUVECs.

RESULTSPPAR alpha, PPAR beta and PPAR gamma mRNA were detected by using RT-PCR in HUVECs. Treatment of HUVECs with PPARalpha and PPAR gamma activators--linolenic acid, linoleic acid, oleic acid and prostaglandin J2 respectively, but not with stearic acid could augment PAI-1 mRNA expression and protein secretion in a concentration-dependent manner. However, the mRNA expressions of 3 subclasses of PPAR with their activators in HUVECs were not changed compared with controls.

CONCLUSIONHUVECs express PPARs. PPARs activators may increase PAI-1 expression in ECs, but the underlying mechanism remains unclear. Although PPARs expression was not enhanced after stimulated by their activators in ECs, the role of functionally active PPARs in regulating PAI-1 expression in ECs needs to be further investigated by using transient gene transfection assay.

Cells, Cultured ; Endothelium, Vascular ; cytology ; metabolism ; Fatty Acids ; pharmacology ; Fetus ; Humans ; Linoleic Acid ; pharmacology ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; Prostaglandin D2 ; analogs & derivatives ; pharmacology ; RNA, Messenger ; genetics ; Receptors, Cytoplasmic and Nuclear ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics ; Umbilical Veins ; cytology ; metabolism ; alpha-Linolenic Acid ; pharmacology