Mechanism of Thapsigargin-induced Apoptosis in MC3T3E1 Osteoblast

Won, Kang-Hee,Kang, Jang-Sook,Chae, Han-Jung,Han, Jo-Il,Kim, Hyung-Ryong 원광대학교 생체재료·매식연구소 1999 원광생체재료·매식 Vol.8 No.1

본 연구는 고농도 칼슘자극에 의한 조골세포주 MC3TE1 세포의 죽음의 단계를 알아보기 위하여 세포내 소기관인 endoplasmic reticulum 의 Ca^++ -ATPase pump를 비가역적으로 억제시키는 물질인 thapsigargin에 의한 조골세포고사 기전을 규명하였다. Thapsigargin은 MC3TE1 세포의 c-Jun N-terminal kinase1 (JNK1)의 활성을 증가시켜 actvating protein-1 (AP-1)의 전사활동을 증가시켰다. Thapsigargin에 의한 MC3TE1의 세포고사 조절기전에 caspase-1과 caspase-3의 활성을 형광기질인 acetyl-YVAD-AMC와 acetyl-DEVD-AMC를 이용하여 관찰한 결과 caspase-3의 활성을 증가시켰으며 또한 nuclear factor-κB (NKκB)의 전사활동을 증가시켰다. 조골세포에서 thapsigargin 에 의한 세포고사 조절기전에 JNK1, caspase-3, protease, AP-1 및 NF-κB의 활성이 관여함을 시사해주고 있다.

Oxygen-derived free radicals and osteoclast differentiation

Chae, Han-Jung,Kim, Jang-Sook,Kim, Hyung-Ryong 원광대학교 생체재료·매식연구소 1977 원광생체재료·매식 Vol.6 No.1

Osteotropic hormones and cytokines are involved in the differentiation of osteoclastic progenitors from hematopoietic stem cells to multinucleated osteoclasts. Recent studies have indicated that oxygen-derived free radicals are implicated in osteoclastic bone resorption. We tested whether superoxide dismutase(SOD), catalase, or hydrogen peroxide induce the generation of osteoclast-like multincleated cells (MNCs). Both SOD and hydrogen peroxide augmented the ability of 1α,25(OH)_2D_3 to induce the generation of osteoclast-like MNCs, but catalase was inhibitory. However, SOD, catalase, or hydrogen peroxide could not increase the generation of osteoclast-like MNCs. These data suggest that oxygen-derived free radicals are involved in the regulation of osteoclast generation.

PGE_2와 DBcAMP가 골조직 세포의 생리적 활성에 미치는 영향

이준기,채한정,강장숙,김형룡 원광대학교 생체재료·매식연구소 1998 원광생체재료·매식 Vol.7 No.2

One of the primary functions for which bones have evolved is to act as structural support. To achieve this goal, bones are remodeling throughout life so that their structure remains optimal for the prevailing mechanical environment. Bone remodeling consists of an initial phase of osteoclastic bone resorption followed by a bone formation period. Prostaglandins are potent regulators of bone formation and bone resorption that can have both stimulatory and inhibitory effects. Elevation of intracellular cAMP is an important intracellular signaling mechanism involved in the regulation of the expression of many proteins. In this study, the author examined whether PGE_2 or DBcAMP affected osteoblastic activation or osteoclastic differentiation in mouse bone marrow cells and osteosarcoma ROS 17/2.8 cells. The effect of PGE_2 and DBcAMP on the cell proliferation was measured by the incorporation of [^3H]-thymidine into DNA. From the result, PGE_2( 0.5-1 ㎍/ml ) and DBcAMP(0.1-0.5 mM) inhibited the [^3H]-thymidine incorporation into DNA. The effect of PGE_2 and DBcAMP on the induction of alkaline phosphatase(ALP) was investigated in ROS 17/2.8 cells cultured in medium containing 0.4% fetal bovine serum. PGE_2 and DBcAMP stimulated ALP activity in the cells. PGE_2 also increased the intracellular cAMPs contents with a maximal effect at 0.5 ㎍/ml. ROS 17/2.8 cells released nitric oxide upon stimulation of PGE2 or DBcAMP with interferon-γ. PGE_2 and DBcAMP increase the phosphorylation level of CREB(cAMP response element binding protein) without any change on the amount of CREB protein. Also, PGE_2 (10^-6 M) and DBcAMP (10^-4 M) significantly increase the generation of osteoclast-like cell in mouse bone marrow cell culture system. In conclusion, the results of this study suggested that PGE_2 and cAMP should appear to be an important regulatory molecule in the processes of bone formation and resorption.

Lidocaine에 의한 경련이 뇌조직에서 c-fos 발현에 미치는 영향

조성범,채한정,강장숙,김형룡 원광대학교 생체재료·매식연구소 1998 원광생체재료·매식 Vol.7 No.1

Recently, a variety of stimuli including convulsion, ischemia and noxious stimuli have been shown to induce the expression of c-fos mRNA in central nervous system (CNS). In this study, c-fos expression in both mRNA and protein (Fos) levels is examined up to 72 hr in brain regions, including hippocampus, piriform cortex, neostriatum, amygdala, dentate gyrus, cerebral cortex, and thalamus during lidocaine-inducced convulsion. Intraperitoneal administration of lidocaine (120 mg/kg) induces generalized convulsion of rats within 10 min and subsequent increase of c-fos expression in both mRNA and Fos protein levels in dentate gyrus, piriform cortex, amygdala, neostriatum, cerebral cortex, hippocampus, and thalamus. A prominent induction of c-fos mRNA and Fos protein in dentate gyrus suggests that dentate gyrus plays an important role in the process of lidocaine-induced convulsion. These results suggest that c-fos may participate in plastic changes of central nervous system associated with stimuli such as seizure activity.

SYNERGISTIC COOPERATION BETWEEN cAMP AND IFN-γ FOR INDUCTION OF NITRIC OXIDE SYNTHESIS IN ROS 17/2.8 OSTEOBLASTS

Chae, Han-Jung,Lee, Jun-Gi,Kang, Jang-Sook,Park, Young-Chul,Park, Joo-Bae,Kim, Hyung-Ryong 원광대학교 생체재료·매식연구소 2000 원광생체재료·매식 Vol.9 No.1

The gas radical, nitric oxide(NO), is a major autacoid regulating cell behavior in the cardioviscular, immune and central nervous systems. Recently, it was shown that it is produced by both the osteoblast and osteoclast and that NO appears to be an important regulatory molecule in the processes of bone remodeling. By measurements of nitrite production, Northen blot analysis, and Western blot analysis, we studied the effects of a membrane-permeable cAMP derivative, DBcAMP and 8-bromo-cAMP, on the expression of inducible nitric oxide synthase (iNOS) mRNA and protein and the synthesis of nitrite in ROS 17/2.8 cells. DBcAMP and 8-bromo-cAMP stimulated NO production and iNOS mRNA and protein expression in IFN-γ-treated ROS 17/2.8 cells in a dose-dependent manner. Compounds that increase intracellular cAMP levels(forskolin, theohylline, PGE_2), all stimulated No production and iNOS mRNA and protein expression in IFN-γ-treated ROS 17/2.8 cells. Sp-cAMPS(a selective activator of cAMP-dependent protein kinase I and II) also increased NO production and iNOS mRNA and protein expression in IFN-γ-treated ROS 17/2.8 cells. N^G - monomethyl-L-arginine, a NOS inhibitor, completely blocked the cAMP-elevating agents-induced NO production IFN-γ-treated ROS 17/2.8 cells. These observations indicate that cAMP is important in considering experiments involving the production of nitric oxide with IFN-γ.

MECHANISM OF MITOGENIC EFFECT OF FLUORIDE ON FETAL RAT OSTEOBLASTIC CELLS : EVIDENCE FOR SHC, GRB2 AND P-CREB-DEPENDENT PATHWAYS

Chae, Han-Jung,Chae, Soo-Wan,Kang, Jang-Sook,Kim, Dae-Eop,Kim, Hyung-Ryong 원광대학교 생체재료·매식연구소 2000 원광생체재료·매식 Vol.9 No.1

Fluoride stimulates bone cell proliferation and nodule formation in fetal rat calvarial osteoblastic cells. In addition, fluoride enhances alkaline phosphatase activity, a marker of osteoblastic differentiation, in a dose-dependent manner. The effects of fluoralumino complex (ALFx) on cell proliferation and differentiation were markedly reduced by tyrosine kinase inhibitor; 1 mM genistein or 1 ㎍/ml herbimycin. It suggests that tyrosine kinase-mediated mitogenic signaling involves a series of protein-protein interactions between tyrosine-phosphorylated receptors, Shc and Grb2, resulting in an AlFx-induced mitogenic effect. The results indicate that AlFx dose-dependently enhances the tyrosine phosphorylation of the adaptor molecule Shc(p52) and its association with Grb2 in the tyrosine kinase mediated pathway. In addition, AlFx decreases the phosphorylation level of CREB without any change on the amount of CREB protein. Taken together, the results suggest that adapter proteins, including Shc and Grb2 of the protein tyrosine kinase cascade are implicated in fluoride-induced mitogenic activity of fetal rat calvarial osteoblastic cells. Furthermore, CREB which passes signals from cAMP to transcriptional factor CRE, modulates the fluroaluminate-induced metabolism of bone cells via a decrease of phosphorylation level.

Signal Transduction of Thapsigargin-induced Apoptosis in Osteoblast

CHAE, H. J.,CHAE, S. W.,WEON, K. H.,KANG, J. S.,KIM, H. R. 원광대학교 생체재료·매식연구소 2000 원광생체재료·매식 Vol.9 No.1

The toxicity of thapsigargin, a selective inhibitor of endoplasmic reticular Ca^2+ -ATPase, was investigated in osteoblasts. We induced apoptosis in murine osteoblastic MC3T3E1 cells by exposure to the thapsigargin. Thapsigargin transiently increased the phosphotransferase activity of c-Jun N-terminal kinases 1(JNK1), which might in turn activate transcriptional activity of activation protein-1(AP-1) we then prepared extracts from thapsigargin-treated MC3T3E1 cells and monitored cleavage of acetyl-YVAD-AMC and acetyl-DEVD-AMC, fluorogenic substrates for caspase 1-like and caspase 3-like proteases, respectively. Thapsigargin significantly increased the proteolytic activity of caspase 3-like proteases, but not the activity of caspase 1-like proteases. Furthermore, thapsigargin increased the transcriptional activity of nuclear factor-κB(NF-κB). These data suggest that thapsigargin-induced apoptosis in osteoblasts may be via activation of JNK1, caspase 3-like family proteases, and transcripitional factors including AP-1 and NF-κB. (Bone 25: 453-458; 1999) ⓒ1999 by Elsevier Science Inc. All rights reserved.