Identification of Genes Modulated by High Extracellular Calcium in Coculture of Mouse Osteoblasts and Bone Marrow Cells by Oligo Chip Assay

Kim, Hyung-Keun,Song, Mina,Jun, ji-Hae,Woo, Kyung-Mi,Kim, Gwan-Shik,Baek, Jeong-Hwa The Korean Academy of Oral Biology 2006 International Journal of Oral Biology Vol.31 No.2

Calcium concentration in the bone resorption lacunae is high and is in the mM concentration range. Both osteoblast and osteoclast have calcium sensing receptor in the cell surface, suggesting the regulatory role of high extracellular calcium in bone merabolism. In vitro, high extracellular calcium stimulated osteoclastogenesis in coculture of mouse osteoblasts and bone marrow cells. Therefore we examined the genes that were commonly regulated by both high extracellular calcium and 1,25(OH)_(2)vitaminD_(3)(VD3) by using mouse oligo 11 K gene chip. In the presence of 10 mM[Ca^(2+)]e or 10 nM VD3, mouse calvarial osteoblasts and bone marrow cells were co-cultured for 4 days when tartrate resistant acid phosphatase-positive multinucleated cells start to appear. Of 11,000 genes examined, the genes commonly regulated both by high extracellular calcium and by VD3 were as follows; 1) the expressions of genes which were osteoclast differentiation markers or were associated with osteoclastogenesis were up-regulated both by high extracellular calcium and by VD3; trap, mmp9, car2, ctsk, ckb, atp6b2, tm7sf4, rab7, 2) several chemokine and chemokine receptor genes such as sdf1, scya2, scyb5, scya6, scya8, scya9, and ccr1 were up-regulated both by high ectracellular calcium and by VD3, 3) the genes such as mmp1b, mmp3 and c3 which possibly stimulate bone resorption by osteoclast, were commonly up-regulated, 4) the gene such as c1q and msr2 which were related with macrophage function, were commonly down-regulated, 5) the genes which possibly stimulate osteoblast differentiation and/or mineralization of extracellular matrix, were commonly down-regulated;slc8a1, admr, plod2, lox, fosb, 6) the genes which possibly suppress osteoblast differentiation and/or mineralization of extracellular matrix, were commonly up-regulated;s100a4, npr3, mme, 7) the genes such as calponin 1 and tgfbi which possibly suppress osteoblast differentiation and/or mineralization of extracellular matrix, were up-regulated by high extracelluar calcium but were down-regulated by VD3. These results suggest that in coculture condition, both high extracellular calcium and VD3 commonly induce osteoclastogenesis but suppress osteoblast differentiation/mineralization by regulating the expression of related genes.

흰쥐 해마박편에서 acetylcholine이 gamma-aminobutyric acid 유리에 미치는 영향에 관한 연구

김익현,김형룡,김관식,정동균 대한구강생물학회 1991 International Journal of Oral Biology Vol.15 No.2

Present study was performed to clarify the effect of acetylcholine on the release of gamma-aminobutyric acid(GABA) employing hippocampal slices. Hippocampal slices (300∼400㎛ thick) were prepared by the method of Kim et al(1988) and pre-equilibrated in Krebs-bicarbonate medium(KBM, pH 7.4) for 1hr at 37℃. Pre-equilibrated slices were incubated in fresh KBM and then potassium(50mM)-containing KBM for 5 min period. Basal and potassium-induced release of GABA were determined from recovered medium by HPLC. After 30min resting period, in the presence of physostigmine(20μM) slices were reincubated in acetylcholine-containing KBM and acetylcholine plus potassium-containing medium consecutively for 5min period each to investigate the effect of acetylcholine on basal or potassium-induced GABA release from hippocampal slices. The observed results were as follows: 1. The release of GABA induced by the first and second 5 min-exposure of 50mM potassium was 107.3±8.2 nmol and 90.6±3.2nmol, respectively. When compared with released amounts of GABA during the corresponding spontaneous periods, these were 4.6 and 4.6-fold increase respectively. 2. Physostigmine(20μM) had no significant effect on the spontaneous release of GABA. 3. Acetylcholine(10-1000μM) increased spontaneous and potassium-induced GABA release in a dose-dependent manner.

흰쥐 해마박편에서 veratrine과 고농도 포타슘자극시 칼슘이온이 gamma-aminobutyric acid 유리에 미치는 영향에 관한 연구 : A role of calcium

강수만,김형룡,김관식,정동균 대한구강생물학회 1991 International Journal of Oral Biology Vol.15 No.2

Present study was performed to clarify the effect of calcium on the release of gamma-aminobutyric acid (GABA) employing hippocampal slices. Hippocampal slices(300-400㎛ thick) were prepared by the method of Kim et al(1988) and pre-equilibrated in Krebs-bicarbonate medium(KBM, pH 7.4) for 1hr at 37℃. In case of veratrine-induced GABA release, pre-equilibrated slices were incubated in fresh KBM and then veratrine (25μM)-containing KBM for 10min period in the presence or absence of 2.5mM Ca^2+. In case of potassium-induced GABA relaese, pre-equilibrated slices were incubated in fresh KBM and then potassium(50mM)-containing KBM for 5min period in the presence or absence of 2.5mM Ca^2+. Basal and veratrine and potassium-induced release of GABA was determined from recovered medium by HPLC. The observed results were as follows: 1. The release of GABA induced by the 10min-exposure of 25μM veratrine and 5min-exposure of 50mM potassium in the presence of 2.5mM Ca^2+ was 228.9±11.2 nmol and 100.1±8.9nmol, respectively. When compared with released amounts of GABA during the corresponding spontaneous periods, these were 6.8 and 4.6-fold increase respectively. 2. The release of GABA induced by the 10min-exposure of 25μM veratrine and 5min-exposure of 50mM potassium in the absence of Ca^2+ was 381.4±30.2 nmol and 55.1±4.1 nmol, respectively. When compared with released amounts of GABA during the corresponding spontaneous periods, these were 11.3 and 2.4-fold increase respectively.

Norepinephrine이 흰쥐 해마박편에서 아미노산 신경전달물질 유리에 미치는 영향에 관한 연구

김성룡,김관식,정동균 대한구강생물학회 1990 International Journal of Oral Biology Vol.14 No.1

Present study was performed to clarify the effect of norepinephrine on the release of amino acid neurotransmitters employing hippocampal slices. Hippocampal slices (300-400㎛ thick) were prepared by the method of Spencer et al.(1976) and pre-equilibrated in Krebs-bicarbonate medium (KBM, pH 7.4) for 1 hr at 37℃. Pre-equilibrated slices were incubated in fresh KBM and then veratrine (25μM)-containing KBM for 10 min period. Basal and veratrine-induced release of GABA and glutamic acid were determined from recovered medium by HPLC. After 30 min resting period, slices were reincubated in norepinephrine-containing KBM and test agent plus veratrine-containing medium consecutively for 10 min period each to investigate the effect of norepinephrine on basal or veratrine-induced amino acid release from hippocampal slices. The observed results were as follows: 1. The release of GABA induced by the first and second 10min-exposure of 25μM veratrine was 246.9±16.82 nmol and 209.9±20.84 nmol, respectively. When compared with released amounts of GABA during the corresponding spontaneous periods, these were 7.3 and 7.3-fold increase respectively. 2. The release of glutamic acid induced by the first and second 10min-exposure of 25μM veratrine was 426.3±63.16 nmol and 377±61.83 nmol, respectively. When compared with released amounts of glutamic acid during the corresponding spontaneous periods, these were 9.9 and 10.2-fold increase respectively. 3. Norepinephrine (1-100μM) increased veratrine-stimulated and spontaneous release of GABA in dose-dependent manner. 4. Norepinephrine (1-100μM) inhibited veratrine-stimulated glutamic acid release in dose-dependent fashion.

Dibutyryl cAMP 와 theophylline이 조골세포군의 alkaline phosphatase에 미치는 영향에 관한 연구

김세원,김관식,정동균 대한구강생물학회 1987 International Journal of Oral Biology Vol.11 No.2

Effects of PGE_2, dibutyryl cAMP (DBcAMP) and theophylline on the acid and alkaline phosphatase of bone cell populations isolated from fetal rat calvaria were studied. Ten fetal rat calvaria of 19th day of gestation were sequentially digested with enzyme solution consisted of 0.1% collagenase, 0.05% trypsin and 0.5mM EDTA for 10, 10, 10, 20 and 20 minutes, and each bone cell population was primarily cultured for 6-7 days. After primary culture, the effects of PGE_2, DBcAMP and theophylline on acid and alkaline phosphatase were studied. The observed results were as follows. 1. Population IV and V of 5 bone cell populations isolated by sequential enzyme digestion were considered to be osteoblast-like cell population. 2. PGE_2 (500ng/ml) increased acid phosphatase in population I and increased alkaline phosphatase in population III, IV and V after 48 hours of culture. 3. DBcAMP (0.5mM) increased alkaline phosphatase in population II, III and V and DBcAMP (0.5mM) plus PGE_2 (500ng/ml) increased alkaline phosphatase in population II, III, IV and V after 24 hours of culture. 4. Theophylline (1mM) increased alkaline phosphatase in population IV and V, and theophylline (1mM) plus PGE_2 (500ng/ml) increased alkaline phosphatase in population III, IV and V after 24 hours of culture.

Propranolol이 흰쥐 해마박편에서 Gamma-aminobutyric acid 및 Glutamic acid유리에 미치는 영향에 관한 연구

김경범,김관식,정동균 대한구강생물학회 1990 International Journal of Oral Biology Vol.14 No.1

Present study was performed to clarify the effect of propranolol on the release of amino acid neurotransmitters employing hippocampal slices. Hippocampal slices (300-400㎛ thick) were prepared by the method of Spencer et al.(1976) and pre-equilibrated in Krebs-bicarbonate medium (KBM, pH 7.4) for 1 hr at 37℃. Pre-equilibrated slices were incubated in fresh KBM and then veratrine (25μM)-containing KBM for 10 min period. Basal and veratrine-induced release of GABA and glutamic acid were determined from recovered medium by HPLC. After 30 min resting period, slices were reincubated in propranolol-containing KBM and propranolol plus veratrine-containing medium consecutively for 10 min period each to investigate the effect of propranolol on basal or veratrine-induced amino acid release from hippocampal slices. The observed results were as follows : 1. The release of GABA induced by the first and second 10min-exposure of 25μM veratrine was 246.9±16.82 nmol and 209.9±20.84 nmol, respectively. When compared with released amounts of GABA during the corresponding spontaneous periods, these were 7.3 and 7.3-fold increase respectively. 2. the release of glutamic acid induced by the first and second 10min-exposure of 25μM veratrine was 426.3±63.16 nmol and 377±61.83 nmol, respectively. When compared with released amounts of glutamic acid during the corresponding spontaneous periods, these were 9.9 and 10.2-fold increase respectively. 3. Propranolol (2-8 mM) inhibited veratrine-stimulated GABA release in dose-dependent manner. 4. Propranolol (2-6 mM) enhanced veratrine-stimulated and spontaneous release of glutamic acid in dose-dependent fashion.

Platelet-Derived Growth Factor가 백서두개관 세포군의 증식 및 교원합성에 미치는 영향

김기수,고성희,백정화,민병무,김관식,정동균 대한구강생물학회 1991 International Journal of Oral Biology Vol.15 No.2

To study the effect of platelet-derived growth factor(PDGF) on the replication and collagen synthesis of rat calvarial cells, five bone cell populations(I-V) were prepared from fetal rat calvaria by sequential enzyme digestion. After primary culture for 6-7 days, each bone cell population was collected and then population Ⅰ and Ⅱ, Ⅳ and Ⅴ were pooled together. And the cells were resuspended at 6-8×10^4 cells/㎝^2 and cultured for 2-3 days. The medium was changed to serum-free medium prior to addition of growth factor. The effect of PDGF on the cell proliferation was measured by the incorporation of [^3H]thymidine into DNA. Protein synthesis was determined by measurement of [^3H]proline incorporation into collagenase-digestible protein(CDP) and noncollagenous protein(NCP) according to the method of Peterkofsky and Die-gelmann(1971). The observed results were as follows. 1. PDGF at 10 ng/㎖ significantly increased the [^3H]thymidine incorporation into DNA in all bone cell populations. 2. PDGF at 30 ng/㎖ significantly increased the synthesis of NCP in population Ⅰ, Ⅱ and Ⅳ, Ⅴ. 3. PDGF had no effect on the synthesis of CDP but percent collagen synthesis was decreased significantly in population Ⅳ, Ⅴ. Taken together, the increase of protein synthesis by PDGF in rat calvarial cells was due to the incraese of NCP synthesis.

흰쥐 두개관 및 간에서 Purine염기의 대사적 운명

김관식,민병무,김세원,이진 대한구강생물학회 1989 International Journal of Oral Biology Vol.13 No.1

As an attempt to clarify the metabolic fate of purine bases in rat calvaria and liver, the activities of xanthine oxidase, guanine aminohydrolase and HGPRT were assayed. The portions of calvaria containing frontal and parietal bone, and liver were dissected from rats. Specimens were assayed for their activities of xanthine oxidase and guanine aminohydrolase in its cytosol and HGPRT in its mitochondrial fraction. Specific activity of hepatic xanthine oxidase with hypoxanthine or xanthine as its substrate was 4.18 or 7.43mU/mg of protein and guanine aminohydrolase was 11.00mU/mg of protein, respectively. In contrast, calvarial xanthine oxidase and guanine aminohydrolase was 1.49 or 1.88 and 0.98, respectively, which were significantly lower than those of hepatic enzymes. Specific activity of calvarial HGPRT was 21.70 and hepatic HGPRT was 13.65mU/mg of protein when hypoxanthine was employed as its substrate while no measurable activity was detected in calvaria when guanine was employed. Comparing to purine-catabolizing enzymes, activity of HGPRT, salvage-enzyme for purine bases, was relatively higher in both tissues and hypoxanthine was utilized preferentially as substrate than guanine. Besides the production of GMP or IMP by HGPRT, concentration of other purine nucleotides such as GMP from guanine or adenylates form hypoxanthine were also increased in hepatic HGPRT assay but no detectable changes in calvaria. Assuming these increments as the consequences of HGPRT activity, it can be deduced that activity of HGPRT in liver would be greater than that of calvaria. Taken together, these results indicated thet ⅰ) in calvaria, most of purine bases would be reutilized by salvage enzymes rather than completely catabolized ⅱ) both of catabolic pathway to final metabolites and salvage pathway for purines would be active as well as de novo synthesis in liver ⅲ) metabolic fate of purine bases varied from tissue to tissue within same species.

Ascorbic acid 가 골조직세포군의 Phosphatase 에 미치는 영향

김상균,김관식,정동균 대한구강생물학회 1987 International Journal of Oral Biology Vol.11 No.2

Several lines of findings suggest that ascorbic acid might influence the function of osteoblasts although no direct evidences were provided. This study was undertaken to investigate the effect of ascorbic acid on bone cells employing 5 fetal rat calvarial cell populations isolated by sequential enzyme digestion. Fetal rat calvaria were treated 5 times consecutively with enzyme mixture of collagenase, trypsin and EDTA for 10, 10, 10, 20 and 20 minutes. Each bone cell population was primarily cultured for 6-7 days and then the effect of ascorbic acid (10 and 100㎍/ml) on the phosphatase level were determined. The observed results were as follows. 1. Population IV and V had characteristics of osteoblast such as high alkaline phosphatase level and low acid phosphatase. 2. Ascorbic acid decreased the acid phosphatase activity of population IV, regardless its concentration while did not affect other cell populations. 3. Alkaline phosphatase activity of population IV was increased significantly by ascorbic acid. 4. Taken together, these results suggest that ascorbic acid may promote the differentiation of osteoblasts and its effect is restricted in osteoblastic population only, not in other type of bone cells.

Lidocaine이 흰쥐 해마박편에서 Gamma-Aminobutyric Acid 유리에 미치는 영향에 관한 연구

김형룡,김관식,정동균 대한구강생물학회 1988 International Journal of Oral Biology Vol.12 No.2

Present study was conducted to clarify the role of GABA in lidocaine-induced convulsion employing hippocampal slice. Hippocampal slices(300-400㎛) were prepared from hippocampal tissue blocks using a glass guide while the tissue was continuously moistened with oxygenated (95% O_2-5% CO_2) Krebs-bicarbonate media (KB) at 37℃. After preparation, the slices were equilibrated for 1hr before biochemical testing in KB. Individual equilibrated slices were transferred to tissue holder, incubated for 5min in fresh oxygenated KB, and then exposed for 5min to 50mM K^+ KB and transferred again to fresh KB for 30min rest period. Each slice served as its own control by exposing it a second time to 50mM K^+ KB for 5min after the 30min rest and a second 5min incubation in fresh KB. After the 30min rest and a second 5min incubation in fresh KB, slices were transferred again to fresh KB containing 1, 2, 3, or 4mM lidocaine, incubated for 10min and then exposed to 50mM K^+ KB+lidocaine solution for 5min. The results were as follows : 1. The release of GABA induced by the first and second 5min exposure of 50mM K^+ was 95.9±17.28nmol and 80.6±13.04nmol, respectively. When compared with released amounts of GABA during the corresponding spontaneous periods, these were 4.7 and 4.9-fold increase respectively and revealed that consecutive K^+─exposure increased GABA release with similar fashion. 2. The concentration of lidocaine necessary for 50 percent inhibition(IC_50) of potassium─induced release of GABA was 1.82mM.