1.The effects of changes in intracellular Ca2+ activity of osteoblast-like cell on fracture healing
Byoung Hyun MIN ; So Ra PARK ; Young Bae KIM ; Chang Kook SUH ; Nam Hyun KIM
The Journal of the Korean Orthopaedic Association 1996;31(4):861-871
Bone formation by osteoblast may be closely related to the increase in intracellular Ca2+ activity of osteoblast. In order to study the effects of changes in Ca2+ activity of osteoblast-like cell on fracture healing, we changed intracellular Ca2+ activity of osteoblast-like cells by vanadate and verapamil. And the process of fracture healing was observed after injection of the treatment osteoblast-like cells into the fracture site by hematoxylin-eosin (H-E) stain and bromodeoxyuridine (BrdU) stain. The results were as follow: 1) The most effective range of concentration which could facilitate bone formation was 10-6 to 10-5 M. 2) H-E stain showed more abundant osteoblast and osteoid tissues, more active mitotic division of osteoblast, and earlier appearance of chondroblast and chondroid tissue, making the maturation of woven bone faster in the vanadate-treated group than in the control group. The opposite was true in the verapamil-treated group compared with the control group. 3) BrdU labeling index showed more active osteoblastic proliferation in the vanadate-treated than in the control group. The opposite was observed in the verapamil-treated group compared with the control group. From these results, the fracture healing appears to be facilitated and decelerated by vanadate which apparently increase intracellular Ca2+ activity of osteoblast and verapamil which decreases it, repectively. Therefore the change of intracellular Ca2+ activity of osteoblast can be considered to be one of fracture healing mechanisms and expected to be applied for clinical purpose.
Bromodeoxyuridine
;
Chondrocytes
;
Fracture Healing
;
Osteoblasts
;
Osteogenesis
;
Vanadates
;
Verapamil
2.Enhanced Radiosensitivity of Tumor Cells Treated with Vanadate in Vitro.
Journal of the Korean Society for Therapeutic Radiology 1994;12(2):129-142
Intracellular ions which have a major role in cellular function have been reported to affect repair of radiation damage. Recently it has been reported that ouabain sensitizes A549 tumor cells hut not CCL-120 normal cells to radiation. Ouabain inhibits the Na+-K+-pump rapidly thus it increases intracellular Na concentration. Vanadate which is distributed extensively in almost all living organisms in known to be a Na+-K+-ATPase inhibitors. This study was performed to see any change in radiosensitivity of tumor cell by vanadate and any role of Na+-K+-ATPase in radiosensitization. Experiments have been carried out by pretreatment with vanadate in human cell line(A549, JMG) and mouse cell line(L1210, spleen). For the cell survival MTT assay was performed for A549 and JMG cell and trypan blue dye exclusion test for L120, and spleen cells. Measurements of Na+-K+-ATPase activity in control, vanadate treated cell, radiation treated cell (9 Gy for A549 and JMG, 2 Gy for L1201, spleen), and combined 10-6 M vanadate and radiation treated cells were done. The results were summarized as follows. 1. L1210 cell was most radiosensitive, and spleen cell and JMG cell were intermediate, and A549 cell was least radiosensitive. 2. Minimum or cytotoxicity was seen with vanadate below concentration of 10-6 M. 3. In A549 cells there was a little change in radiosensitivity with treatment of vanadate. However radiation sensitization was shown in low dose level of radiation i. E. 2-Gy. In JMG cells no change in radiosensitivity was noted. Both L1210 and spleen cell had radiosensitization but change was greater in tumor cell. 4. Na+-K+-ATPase activity was inhibited significantly in tumor cell by treatment of vanadate. 5. Radiation itself inhibited Na+-K+-ATPase activity of tumor cell with high Na+- K+-ATPase concention. Increase in radiosensitivity by vanadate was closely associated with original Na+-K+-ATPase contents. From the above results vanadate had little cytotoxicity and it sensitized tumor cells to radiation. Inhibitory effect of vanadate on Na+-K+-ATPase activity might be one of the contributing factors for radiosensitization to tumor cells which has greater enzyme activity than that of normal cell. It was suggested vanadate could be used as a potential radiosensitizer for tumor cells.
Animals
;
Cell Survival
;
Humans
;
Ions
;
Mice
;
Ouabain
;
Radiation Tolerance*
;
Spleen
;
Trypan Blue
;
Vanadates*
3.The effect of sodium fluoride and sodium orthovanadate on osteoblastic cell line MC3T3-E1 cells.
Korean Journal of Orthodontics 1991;21(1):97-111
It is the aim of this study to investigate the effects of sodium fluoride and sodium orthovanadate upon the proliferation and activity of the osteoblast (MC3T3-E1 cells). MC3T3-E1 cells were cultured in alpha-MEM containing 10% FBS and various concentration of sodium fluoride and sodium orthovanadate was appended to serum free media. DNA synthesis was examined through the [3H] thymidine incorporation into DNA. Collagen synthesis was examined through the [3H] proline incorporation into collagenase digestible protein and noncollagen protein. The following results were drawn; 1. Sodium fluoride stimulated the DNA synthesis of osteoblast significantly in dose-dependent manner within the concentration from 2micrometer to 10micrometer (P<0.005). 2. Sodium orthovanadate stimulated the DNA synthesis of osteoblast significantly in dosedependent manner within the concentration from 2micrometer to 8micrometer, however showed diminution at 10micrometer (P<0.001). 3. Sodium fluoride and sodium orthovanadate stimulated the percent collagen synthesis of osteoblast significantly in dose-dependent manner within the concentration from 5micrometer to 10micrometer (P<0.001). 4. Sodium fluoride and sodium orthovanadate stimulated the non collagen synthesis of osteoblast significantly in dose-dependent manner within the concentration from 5micrometer to 10micrometer (P<0.001). In conclusion, sodium fluoride and sodium orthovanadate stimulate the proliferation and activity of osteoblast by stimulation of DNA synthesis and collagen and noncollagen synthesis in osteoblast.
Cell Line*
;
Collagen
;
Collagenases
;
Culture Media, Serum-Free
;
DNA
;
Osteoblasts*
;
Proline
;
Sodium Fluoride*
;
Sodium*
;
Thymidine
;
Vanadates*
4.The effects of vanadium oxide & sodium orthovanadate on murin osteoblast-like (MC3T3-E1) cells.
Korean Journal of Orthodontics 1994;24(1):17-35
Vanadium is an essential trace element but has not been identified with a specific biogical role. To study the direct effects of vanadium on osteoblast, we incubated murin osteoblast-like (MC3T3-E1) cells with various concentration of vanadium oxide & sodium orthovanadate. This study was designed to investigate the effect of vanadium on DNA synthesis, alkaline phosphatase (ALP) activity, cAMP formation responsive to parathormone(PTH) and type I alpha 2 collagen ribonucleic acid (mRNA) level in murin osteoblast-like (MC3T3-E1) cells. The cells were cultured in a -minimal essential medium(alpha -MEM) supplemented with 10% fetal bovine serum (FBS) and then changed to 0.1% FBS with various concentration of vanadium oxide & sodium orthovanadate. Quiescent cultured MC3T3-E1 cells incubated for 24 hours with 2,5,10,15,20 micrometer vanadium oxide incorporated [3H]Thymidine; every concentration showed increases in [3H]Thymidine incorporations dose dependant manner, the greatest response occurred at 20micrometer. Quiescent cultured MC3T3- E1 cells incubated for 3days with 2,5,10,15,20 micrometer vanadium oxide, for 2 days with sodium orthovanadate and alkaline phosphatase was assayed with disodium phenyl phosphate as substrate. Vanadium oxide increased the alkaline phosphatase content in MC3T3- E1 cells at 2 micrometer & 6micrometer; the greatest response occurred at 2micrometer. But decreased at other content. sodium orthovanadate increased alkaline phosphatase content in MC3T3-E1 cells at all concentration ; the greatest response occurred at 4micrometer. Quiescent cultured MC3T3- E1 cells incubated for 3days with 5,10micrometer vanadium oxide, with 5,8micrometer sodium orthovanadate and cAMP formation was measured by Radioimmunoassay(RIA). Vanadium oxide & sodium orthovanadate showed the tendency of inhibitory effects on cAMP responsiveness to PTH in MC3T3-E1 cells. Quiescent cultured MC3T3-E1 cells incubated for 24hours with 10,20micrometer vanadium oxide, with 5,10micrometer sodium orthovanadate and Type I alpha2 collagen ribonucleic acid (mRNA) expression was studied by Northern blot analysis. Northern blot analysis of vanadium oxide treated cells showed decreasing effects 0& sodium orthovanadate revealed increasing effects in type I &2 collagen ribonucleic acid (mRNA) level.
Alkaline Phosphatase
;
Blotting, Northern
;
Collagen
;
DNA
;
Osteoblasts
;
RNA
;
Sodium*
;
Vanadates*
;
Vanadium*
5.Effects of sodium vanadate on contractility of vascular smooth muscle.
Young Kwang YOON ; Chang Hyun MOON ; Young Ho LEE ; Bok Soon KANG ; Doo Hee KANG
Korean Circulation Journal 1993;23(1):104-116
The present study was intended to examine the effect of sodium vanadate on contractility of vascular smooth muscle. Aortic ring preparations were made from the rabbit thoracic aorta and endothelial cells were removed from the ring. The contractility of the aortic ring was measured under various conditions. The results were summarized as follows; 1) Sodium vanadate induced contraction of vascular smooth muscle in a dose-dependent fashion. 2) The contractile effects were not blocked by treatments with adrenergic blocking agent(phentolamine) and indomethacin, indicating the direct action of the drug on vascular smooth muscle. 3) In the presence of ouabain, Na(+)-K(+)-ATPase inhibitor, sodium vanadate still increased the contractility of vascular smooth muscle. 4) Treatment with 4.4'-diisothiocyanostilbene-2.2'-disulfonic acid(DIDS) blocked completely the contractile effects of sodium vanadate. 5) In the presence of verapamil, lanthanum and ryanodine, the contractility of the vascular smooth muscle by sodium vanadate was decreased. From the above results. it was suggested that sodium vanadate acts directly on vascular smooth muscle and causes contraction. It was probably due to inhibition of Ca(++)-ATPase in plasma membrane as well as increasing the release of Ca(++) from sarcoplasmic reticulum and Ca(++) influx across the plasma membrane, but not inhibition of Na(+)-K(+)-ATPase.
Aorta, Thoracic
;
Cell Membrane
;
Endothelial Cells
;
Indomethacin
;
Lanthanum
;
Muscle, Smooth, Vascular*
;
Ouabain
;
Ryanodine
;
Sarcoplasmic Reticulum
;
Sodium*
;
Vanadates*
;
Verapamil
6.Effect of Orthovanadate on Collagen and Fibronectin Synthesis, and Alkaline Phosphatase Activity in MC3E3-E1 Osteoblast cells.
Shin Yoon KIM ; Joo Chul IHN ; Je Yong CHOI ; Jong Chul AHN
The Journal of the Korean Orthopaedic Association 2003;38(2):133-141
PURPOSE: The purpose: of this study was to know the effect of inhibition of protein dephosphorylation on the synthesis of collagen and fibronectin (FN), alkaline phosphatase (ALP) activity, and the formation of bone nodule in MC3T3-E1 osteoblasts using orthovanadate (OVA) which is a potent protein tyrosine phosphatase (PTPases) inhibitor. MATERIALS AND METHODS: The synthesis of collagen, noncollagenous protein (NCP), and percent collagen in MC3T3-E1osteoblasts with or without OVA treatment according to concentration and time sequence was determined by incorporation of [3 H]-proline, synthesis of FN by [35 S] methionine incorproated immunoprecipitation after treatment with 100 M OVA for 24 hours, mRNA expression of collagen and FN by Northern blotting, activity of ALP by spectrophotometric method, and formation of bone nodule by staining method. RESULTS: OVA increased collagen and NCP synthesis concentration dependently, until 12 hours in short-time culture, and time dependently through the differentiation until 29 days, however, there was no significant effect on the percent collagen production. OVA increased percent collagen synthesis significantly at 6 hours, and decreased in a long time culture. Total FN synthesis and FN synthesis in cell layer were increased by OVA, however, FN synthesis in medium was not changed. OVA decreased collagen mRNA level dose-dependently and increased the steady-state level of FN mRNA. OVA inhibited activity of ALP in both short and long-time culture. OVA inhibited bone nodule formation in MC3T3-E1 osteoblasts. CONCLUSION: These results indicate that the inhibition of PTPase by OVA increased the synthesis of collagen, FN, and decreased ALP activity and it resulted in the inhibition of bone formation in MC3T3-E1 osteoblast cells.
Alkaline Phosphatase*
;
Blotting, Northern
;
Collagen*
;
Fibronectins*
;
Immunoprecipitation
;
Methionine
;
Osteoblasts*
;
Osteogenesis
;
Ovum
;
Protein Tyrosine Phosphatases
;
RNA, Messenger
;
Vanadates*
7.Thyrotropin Suppresses INF-r Mediated Gene Expression by Inhibiting Signal Transducer and Activation of Transcription 1(STAT1) Activity in FRTL-5 Cells.
Min Ho SONG ; Young Kun KIM ; Heung Kyu RO ; Eun Shin PARK ; Soon Hee YOO ; Ho KIM ; Kang Wook LEE ; Hee Jung HAN ; Won Chan JOO ; Jin Ho WON ; Kyu LIM ; Oh Yoo KWON
Journal of Korean Society of Endocrinology 1998;13(4):536-553
BACKGROUND: The proinflammatory cytokine, IFN-y has been shown to exert pleiotropic effects in a variety of pathophysiologic conditions in autoimmune thyroid disease. The thyrocyte response to IFN-y is mediated two distinct classes of proteins, Janus kinases(Jakl and Jak2) and Signal Transducers and Activation of Transcription(STATl). The activation of STAT 1 is involved in the regulation of many interferon stimulated genes, such as MHC class II, intercellular adhesion molecules-1(ICAM-1) and MHC class II transactivator(CIITA) after the binding to the GASgFN- pactivated site) of the gene promoters. Recently we found TSH/forskolin inhibits IFN-y stimulated maximal expression of ICAM-1 in FRTL-5 cell. IFN-y action is localized between -175 bp and -97 bp from the start of translation of ICAM-1 gene which contains regulatory elements known to be involved in IFN-y action in other eukaryotic cells, palindromic IFN-y activated site(GAS)(5-TTTCCGGGAAA-3) which could bind STAT1, STAT3, STAT5, STAT6. Furthermore, the addition of TSH and forskolin causes a decrease in ICAM-1 promoter activity and its action was localized in GAS. These findings suggested TSH/cAMP signaling pathways downregulate IFN-y activated Janus kinase-STAT signaling path. We wanted to explore the possible involvement of elevated cAMP in the negative regulation of IFN-y induced STAT1 activation in thyroid cells. METHOD: We made several 5-deletion constructs of rat ICAM-1 promoter and analyzed the promoter activities by measuring the luciferase activity after tranfection into FRTL-5 cells. The protein/DNA complex was measured by electrophoretic mobility shift analysis using labeled oligonucleotide. We checked the level of total and phosphorylated STATl protein by immunoblot analysis using specific antibodies. RESULTS: Stimulation of IFN-y in FRTL-5 cells resulted in rapid activation of STATl/DNA binding activity, which was apparent after several minute of stimulation, maintains its activity until 48 h. Incubation of cells with TSH result in suppression of IFN-p mediated STAT1/DNA binding activity throughout the time course of activation by IFN-y. Addition of TSH into 5H maintained FRTL-5 cells did not change the total amount of latent STAT1 amount and also not affect IFN-y mediated production of total STAT1 until 4 h. IFN-y(100 U/mL) rapidly induced phosphorylation of STAT1 within 30 min. and maintained its level without significant change until 48 hours. Cells treated with TSH dramatically lowered the level of IFN-y induced production and phosphorylation of STAT1 after 12 h, 24 h, 36 h, and 48 h but TSH had no effect on the level of phosphorylated STATl within 4 h after IFN-y stimulation. The proteasome inhibitor, MG132 and phosphatase inhibitor, sodium orthovanadate did not block the TSH or forskolin mediated downregulation of phosphorylated STAT1. CONCLUSION: These results indicate a regulatory mechanism which TSH signaling can modulate the prolonged activation of Jak/Stat by IFN-y. We identified one of mechanisms related to TSH mediated negative suppression of the ICAM-1 gene; TSH/cAMP signaling pathways downregulate the cytokine activated Janus kinase-STAT signaling path.
Animals
;
Antibodies
;
Colforsin
;
Down-Regulation
;
Eukaryotic Cells
;
Gene Expression*
;
Intercellular Adhesion Molecule-1
;
Interferons
;
Luciferases
;
Phosphorylation
;
Proteasome Inhibitors
;
Rats
;
Sodium
;
Thyroid Diseases
;
Thyroid Gland
;
Thyrotropin*
;
Transducers*
;
Vanadates
8.Triptolide Inhibits the Proliferation of Immortalized HT22 Hippocampal Cells Via Persistent Activation of Extracellular Signal-Regulated Kinase-1/2 by Down-Regulating Mitogen-Activated Protein Kinase Phosphatase-1 Expression.
Hee Sang KOO ; Sung Don KANG ; Ju Hwan LEE ; Nam Ho KIM ; Hun Taeg CHUNG ; Hyun Ock PAE
Journal of Korean Neurosurgical Society 2009;46(4):389-396
OBJECTIVE: Triptolide (TP) has been reported to suppress the expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), of which main function is to inactivate the extracellular signal-regulated kinase-1/2 (ERK-1/2), the p38 MAPK and the c-Jun N-terminal kinase-1/2 (JNK-1/2), and to exert antiproliferative and pro-apoptotic activities. However, the mechanisms underlying antiproliferative and pro-apoptotic activities of TP are not fully understood. The purpose of this study was to examine whether the down-regulation of MKP-1 expression by TP would account for antiproliferative activity of TP in immortalized HT22 hippocampal cells. METHODS: MKP-1 expression and MAPK phosphorylation were analyzed by Western blot. Cell proliferation was assessed by 3H-thymidine incorporation. Small interfering RNA (siRNA) against MKP-1, vanadate (a phosphatase inhibitor), U0126 (a specific inhibitor for ERK-1/2), SB203580 (a specific inhibitor for p38 MAPK), and SP600125 (a specific inhibitor for JNK-1/2) were employed to evaluate a possible mechanism of antiproliferative action of TP. RESULTS: At its non-cytotoxic dose, TP suppressed MKP-1 expression, reduced cell growth, and induced persistent ERK-1/2 activation. Similar growth inhibition and ERK-1/2 activation were observed when MKP-1 expression was blocked by MKP-1 siRNA and its activity was inhibited by vanadate. The antiproliferative effects of TP, MKP-1 siRNA, and vanadate were significantly abolished by U0126, but not by SB203580 or SP600125. CONCLUSION: Our findings suggest that TP inhibits the growth of immortalized HT22 hippocampal cells via persistent ERK-1/2 activation by suppressing MKP-1 expression. Additionally, this study provides evidence supporting that MKP-1 may play an important role in regulation of neuronal cell growth.
Anthracenes
;
Blotting, Western
;
Butadienes
;
Cell Proliferation
;
Diterpenes
;
Down-Regulation
;
Epoxy Compounds
;
Imidazoles
;
Neurons
;
Nitriles
;
p38 Mitogen-Activated Protein Kinases
;
Phenanthrenes
;
Phosphorylation
;
Protein Kinases
;
Pyridines
;
RNA, Small Interfering
;
Vanadates
9.Effects of vanadate on vascular contractility and membrane potential in the rabbit aorta.
Sang Man CHUNG ; Duck Sun AHN ; Hye Sun SEOK ; Yong JEONG ; Bok Soom KANG
Yonsei Medical Journal 1992;33(1):14-23
Isolated rabbit aortic ring with intact endothelial cell preparations precontracted with NE (10(-7) M) were relaxed by vanadate in a dose dependent manner (from 0.2 to 2 mM). Application of vanadate and ACh during the tonic phase of high K+(100 mM)-induced contraction showed a slight relaxation in contrast to that in NE-induced contraction, but sodium nitroprusside (10 microM) more effectively relaxed the aortic ring preparations in high K+ contraction than that of vanadate. Vanadate-induced relaxation in NE-contracted aortic rings was reversed by application of BaCl2 (50 microM) or glibenclamide (10 microM). Furthermore, Vanadate hyperpolarized membrane potential of smooth muscle cells in endothelium-intact aortic strips and this effect was abolished by application of glibenclamide. The above results suggest that vanadate release EDHF (Endothelium-Derived Hyperpolarizing Factor), in addition to EDRF (Endothelium-Derived Relaxing Factor) from endothelial cell. This EDHF hyperpolarize the smooth muscle cell membrane potential via opening of the ATP-sensitive K+ channel and close a voltage dependent Ca++ channel. So it is suggested that the vanadate-induced relaxation of rabbit thoracic aortic rings may be due to the combined effects of EDRF and EDHF.
Animal
;
Aorta/drug effects/physiology
;
In Vitro
;
Membrane Potentials/drug effects
;
Potassium/pharmacology
;
Potassium Channels/physiology
;
Rabbits
;
Support, Non-U.S. Gov't
;
Tetraethylammonium Compounds/pharmacology
;
Vanadates/*pharmacology
;
Vasodilation/*drug effects
10.Insulin-dependent Stimulation of a Subtype of p38Map Kinases and Its Role in Insulin's Antiapoptotic Activity.
Shin Hae KANG ; Ji Hoon KANG ; Hee Kyoung KANG ; Dae Ho LEE ; Young Ki LEE ; Deok Bae PARK
Journal of Korean Society of Endocrinology 2004;19(4):358-368
BACKGROUND: The p38 mitogen-activated protein kinases (p38Map kinases) are a family of prolinedirected serine/threonine kinases. At least four isoforms of p38Map kinases have been identified; however, their physiological significances remain to be understood. Recently, the role of p38Map kinase in insulin-stimulated glucose uptake has been suggested. The present study aimed to investigate which isoform(s) were responsive to insulin stimulation. In addition, the activities of p38 Map kinase isoforms that may participate in the insulin's antiapoptotic function in CHO-IR cells were also determined. METHODS: Chinese hamster ovary cells, expressing wild- or mutated human insulin receptors (CHO-IR cells), were used to investigate whether insulin can stimulate any of the isoform(s) of the p38Map kinases. The p38Map kinase activity was determined by measuring the degree of 32P-labelling of ATF-2 protein, a specific substrate of p38Map kinase. A DNA laddering assay was performed to examine the degree of apoptosis and a RT-PCR analysis to determine which isoform(s) of the p38Map kinases were expressed in response to insulin. RESULTS: p38Map kinase activation by insulin was sharply suppressed in only the CHO-IR/A1018K cells, which lack the intrinsic tyrosine kinase activity of insulin receptors. Insulin stimulation of p38Map kinase was insensitive to SB203580, an inhibitor of the alpha(alpha)-and beta(beta)-isoforms of p38Map kinases. Moreover, orthovanadate, known as a specific stimulator of the gamma(gamma)-and delta(delta-) isoforms, stimulated the p38Map kinase activity in CHO-IR cells. Insulin increased the degree of mRNA expression of the delta-isoform, but not that of the alpha-isoform p38Map kinase. Interestingly, PD98059, an inhibitor of ERK, suppressed p38Map kinase stimulation, as well as the antiapoptotic protection of cells by insulin. As insulin was found to still protect ERK-lacking cells (CHO-IR/ SOS) from apoptosis, any substantial role(s) of ERK might be excluded. CONCLUSION: Our data suggest that insulin may stimulate the activity and expression of the-isoform of p38Map kinase in a MEK1/2-dependent manner. The involvement of the delta-isoform of p38Map kinase in insulin's antiapoptotic protection was also suggested, but remains to be investigated further to clarify the nature of its mechanism of action
Animals
;
Apoptosis
;
Cricetinae
;
Cricetulus
;
DNA
;
Female
;
Glucose
;
Humans
;
Insulin
;
Ovary
;
p38 Mitogen-Activated Protein Kinases
;
Phosphotransferases*
;
Protein Isoforms
;
Protein-Tyrosine Kinases
;
Receptor, Insulin
;
RNA, Messenger
;
Vanadates