• International Journal of Oral Biology
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• 제36권4호
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• pp.173-178
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• 2011

Tumor necrosis factor alpha ($TNF{\alpha}$) is a multifunctional cytokine that is elevated in inflammatory diseases such as atherosclerosis, diabetes and rheumatoid arthritis. Recent evidence has suggested that ${\beta}2$ adrenergic receptor (${\beta}2AR$) activation in osteoblasts suppresses osteogenic activity. In the present study, we explored whether $TNF{\alpha}$ modulates ${\beta}AR$ expression in osteoblastic cells and whether this regulation is associated with the inhibition of osteoblast differentiation by $TNF{\alpha}$. In the experiments, we used C2C12 cells, MC3T3-E1 cells and primary cultured mouse bone marrow stromal cells. Among the three subtypes of ${\beta}AR$, ${\beta}2$ and ${\beta}3AR$ were found in our analysis to be upregulated by $TNF{\alpha}$. Moreover, isoproterenol-induced cAMP production was observed to be significantly enhanced in $TNF{\alpha}$-primed C2C12 cells, indicating that $TNF{\alpha}$ enhances ${\beta}2AR$ signaling in osteoblasts. $TNF{\alpha}$ was further found in C2C12 cells to suppress bone morphogenetic protein 2-induced alkaline phosphatase (ALP) activity and the expression of osteogenic marker genes including Runx2, ALP and osteocalcin. Propranolol, a ${\beta}2AR$ antagonist, attenuated this $TNF{\alpha}$ suppression of osteogenic differentiation. $TNF{\alpha}$ increased the expression of receptor activator of NF-${\kappa}B$ ligand (RANKL), an essential osteoclastogenic factor, in C2C12 cells which was again blocked by propranolol. In summary, our data show that $TNF{\alpha}$ increases ${\beta}2AR$ expression in osteoblasts and that a blockade of ${\beta}2AR$ attenuates the suppression of osteogenic differentiation and stimulation of RANKL expression by $TNF{\alpha}$. These findings imply that a crosstalk between $TNF{\alpha}$ and ${\beta}2AR$ signaling pathways might occur in osteoblasts to modulate their function.

• 대한의생명과학회지
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• 제17권3호
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• pp.217-223
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• 2011

${\beta}$-sitosterol glucoside exists in a variety of plants and have anti-tumor, anti-microbial, and immunomodulatory activities. Mast cells and eosinophils play important roles in a variety of inflammatory diseases, specifically asthma and atopic dermatitis. In the present study, we used lactose-${\beta}$-sitosterol (L-BS) and investigated the effect of L-BS on inflammatory responses of the human mast cell line, HMC-1 and the human eosinophilic leukemia cell line, EoL-1. In HMC-1 cells, L-BS significantly inhibited cell migration in response to stem cell factor without cytotoxicity. However, the mRNA expression of CC chemokine receptors (CCRs), including CCR1-5, were not altered after L-BS treatment in HMC-1 cells. LPS-induced IL-4 production was also suppressed by L-BS in a dose-dependent manner. In EoL-1 cells, the concentration ranging from 0.1 ${\mu}M$ to 10 ${\mu}M$ of L-BS had no cytotoxicity and had no effect on mRNA expression of major protein-mediators derived from activated eosinophils. However, 100 ${\mu}M$ of L-BS induced the apoptosis of EoL-1 cells in a time-dependent manner. This finding indicates the possibility of L-BS as a potential therapeutic molecule in inflammatory diseases and may contribute to the need to improve current therapeutic drugs.

• Asian-Australasian Journal of Animal Sciences
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• 제22권5호
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• pp.618-625
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• 2009

The objective of this work was to study the direct effects of daidzein on steroidogenesis in cultured mouse Leydig cells. Adult mouse Leydig cells were purified by Percoll gradient centrifugation, and the cell purity was determined using a $3{\beta}$-hydroxysteroid dehydrogenase ($3{\beta}$-HSD) staining method. The purified Leydig cells were exposed to different concentrations ($10^{-7}$ M to $10^{-4}$ M) of daidzein for 24 h under basal and human chorionic gonadotropin (hCG)-stimulated conditions. The cell viability and testosterone production were determined, and the related mechanisms of daidzein action were also evaluated using the estrogen receptor antagonist ICI 182,780 and measuring the mRNA levels of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and $3{\beta}$-HSD-1 involved in testosterone biosynthesis. The results revealed that daidzein did not influence cell viability. Daidzein increased both basal and hCG-stimulated testosterone production in a dose-dependent manner, and this effect was statistically significant at concentrations of $10^{-5}$ M and $10^{-4}$ M daidzein (p<0.05). ICI 182,780 had no influence on daidzein action. RTPCR results revealed that $10^{-5}$ M and $10^{-4}$ M daidzein did not exert any obvious influence on the mRNA level of P450scc in Leydig cells. However, in the presence of hCG, these concentrations of daidzein significantly increased the StAR and $3{\beta}$-HSD-1 mRNA levels (p<0.05), but in the absence of hCG, only $10^{-5}$ M and $10^{-4}$ M daidzein up-regulated the StAR and $3{\beta}$-HSD-1 mRNA expression (p<0.05), respectively. These results suggest that daidzein has direct effect on Leydig cells. Daidzein-induced increase of testosterone production is probably not mediated by the estrogen receptor but correlates with the increased mRNA levels of StAR and $3{\beta}$-HSD-1.

• Molecules and Cells
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• 제41권3호
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• pp.234-243
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• 2018

In recent years, the interest towards the relationship between incretins and bone has been increasing. Previous studies have suggested that glucagon-like peptide-1 (GLP-1) and its receptor agonists exert beneficial anabolic influence on skeletal metabolism, such as promoting proliferation and differentiation of osteoblasts via entero-osseous-axis. However, little is known regarding the effects of GLP-1 on osteoblast apoptosis and the underlying mechanisms involved. Thus, in the present study, we investigated the effects of liraglutide, a glucagon-like peptide-1 receptor agonist, on apoptosis of murine MC3T3-E1 osteoblastic cells. We confirmed the presence of GLP-1 receptor (GLP-1R) in MC3T3-E1 cells. Our data demonstrated that liraglutide inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as detected by Annexin V/PI and Hoechst 33258 staining and ELISA assays. Moreover, liraglutide upregulated Bcl-2 expression and downregulated Bax expression and caspase-3 activity at intermediate concentration (100 nM) for maximum effect. Further study suggested that liraglutide stimulated the phosphorylation of AKT and enhanced cAMP level, along with decreased phosphorylation of $GSK3{\beta}$, increased ${\beta}-catenin$ phosphorylation at Ser675 site and upregulated nuclear ${\beta}-catenin$ content and transcriptional activity. Pretreatment of cells with the PI3K inhibitor LY294002, PKA inhibitor H89, and siRNAs GLP-1R, ${\beta}-catenin$ abrogated the liraglutide-induced activation of cAMP, AKT, ${\beta}-catenin$, respectively. In conclusion, these findings illustrate that activation of GLP-1 receptor by liraglutide inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation through $cAMP/PKA/{\beta}-catenin$ and $PI3K/Akt/GSK3{\beta}$ signaling pathways.

• 대한치과교정학회지
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• 제39권4호
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• pp.248-256
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• 2009

이 연구의 목적은 배양된 사람 치주인대 세포에서 파골세포의 형성에 관련된 물질을 합성, 분비할 수 있는지를 알아보고 압박력이 M-CSF, IL-$1{\beta}$, RANKL 및 OPG mRNA의 발현에 미치는 영향을 알아보고자 하였다. 교정치료를 목적으로 발치된 소구치에서 얻은 치주인대세포를 배양한 후 다양한 크기(0.5, 1.0, 2.0, 3.0, $4.0\;g/cm^2$)의 기계적 자극을 다양한 기간(0.5, 1.5, 6, 24, 48 hours) 동안 적용하고, M-CSF, IL-$1{\beta}$, RANKL, OPG mRNA 발현양의 변화를 검사하였다. 각각의 실험군에서 얻어진 mRNA에 대해 역전사 중합효소 연쇄반응검사를 시행하였다. 검사 결과 압박력은 사람 치주인대 세포에서 M-CSF mRNA를 발현시켰으며 M-CSF, IL-$1{\beta}$, RANKL mRNA의 발현양은 자극의 크기와 기간에 따라 증가하였다. 그러나 압박력은 사람 치주인대 세포에서 OPG mRNA의 발현양에 영향을 미치지 않는 것으로 나타났다. 이상의 결과는 기계적 자극이 치주인대 세포에서 M-CSF, IL-$1{\beta}$, RANKL mRNA의 발현양을 조절함으로 파골세포의 분화에 영향을 미칠 수 있음을 시사한다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권18호
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• pp.8177-8186
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• 2016

The present study was aimed at determining the effects of alkylating and oxidative stress inducing agents on a newly identified variant of DNA polymerase beta ($pol{\beta}{\Delta}_{208-304}$) specific for ovarian cancer. $Pol{\beta}{\Delta}_{208-304}$ has a deletion of exons 11-13 which lie in the catalytic part of enzyme. We compared the effect of these chemicals on HeLa cells and HeLa cells stably transfected with this variant cloned into in pcDNAI/neo vector by MTT, colony forming and apoptosis assays. $Pol{\beta}{\Delta}_{208-304}$ cells exhibited greater sensitivity to an alkylating agent and less sensitivity towards $H_2O_2$ and UV when compared with HeLa cells alone. It has been shown that cell death in $Pol{\beta}{\Delta}_{208-304}$ transfected HeLa cells is mediated by the caspase 9 cascade. Exon 11 has nucleotidyl selection activity, while exons 12 and 13 have dNTP selection activity. Hence deletion of this part may affect polymerizing activity although single strand binding and double strand binding activity may remain same. The lack of this part may adversely affect catalytic activity of DNA polymerase beta so that the variant may act as a dominant negative mutant. This would represent clinical significance if translated into a clinical setting because resistance to radiation or chemotherapy during the relapse of the disease could be potentially overcome by this approach.

• Clinical and Experimental Reproductive Medicine
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• 제35권1호
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• pp.49-60
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• 2008

목 적: 자궁내막조직에서 분리한 상피세포와 기질세포를 삼차원 공배양을 통한 탈락막화 유도에서 성호르몬과 TGF-${\beta}1$의 역할을 알아보고 2-세포기 생쥐배아와 탈락막화가 유도된 자궁내막세포와의 공배양을 통하여 포배형성율, 부화율, 포배기배아의 내세포괴와 영양막세포수 및 부착율을 알아보기 위해 시행되었다. 연구방법: 인간 자궁내막조직에서 분리된 기질세포와 상피세포의 표지인자인 cytokeratin과 vimentin에 대한 면역조직 화학염색을 실시하여 분리를 확인하였으며, 성호르몬 우세환경 (progesterone, estrogen)에서 분리된 세포를 단일배양 혹은 3차원 공배양을 통하여 RT-PCR법으로 TGF-${\beta}1$, 수용체-1, -2, integrin-${\beta}3$, prolactin의 발현을 조사하였다. 배양액군을 대조군으로 하여 2-세포기 생쥐배아와 탈락막화 유도와 유도하지 않은 인간 자궁내막세포와의 공배양을 통하여 포배형성율, 부화율, 부착율과 부화된 포배의 영양막세포와 내세포괴수를 비교하였다. 결 과: 상피세포 표지인자인 cytokeratin과 기질세포 표지인자인 vimentin을 이용하여 면역조직화학염색을 한 결과 각각 95% 이상에서 양성반응을 나타내어 자궁내막조직으로부터 상피세포와 기질세포가 성공적으로 분리되었음을 확인하였다. 분리된 상피세포와 기질세포를 단일배양에서는 성호르몬의 조건에 관계없이 TGF-${\beta}1$과 수용체 type-1, type-2, integrin-${\beta}3$, prolactin mRNA가 발현되지 않았다. 공배양에서는 progesterone 우세환경일 경우 TGF-${\beta}1$ 수용체 type-2를 제외한 모든 mRNA가 발현하였으나 estrogen 우세환경에서는 TGF-${\beta}1$ 수용체 type-2와 prolactin이 발현되지 않았다. 2-세포기 생쥐배아를 배양액군, 비탈락막군 및 탈락막군으로 나누어 공배양하였을 때 포배기 발달율은 차이가 없었으나 부화율 (92%)과 부착율 (82%)은 탈락막군이 유의하게 높았으며 (p<0.05), 비탈락막군의 공배양에서 다수의 영양막세포가 투명대를 완전히 빠져나오지 않은 상태로 부착한 비정상형태를 보였다. 부화된 생쥐 포배기배아의 내세포괴수는 탈락막화에 관계없이 공배양한 포배의 내세포괴수가 유의하게 많았으며 (p<0.05), 영양막세포수는 탈락막군에서 배양액군과 비탈락막군보다 유의하게 많았다 (p<0.05). 결 론: 자궁내막조직에서 상피세포와 기질세포를 분리하여 다시 삼차원적 공배양을 통하여 progesterone (100 nM), estrogen (1 nM)과 TGF-${\beta}1$ (10 ng/ml)을 첨가하면 체외에서 탈락막화를 유도할 수 있으며, 탈락막화를 유도한 자궁내막 세포와 2-세포기 생쥐배아를 공배양하였을 때 탈락막화가 부화율, 부착율 및 영양막세포수에 유효한 영향을 미치는 것을 알 수 있었다.

• Molecules and Cells
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• 제39권11호
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• pp.821-826
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• 2016

The ${\beta}$-catenin functions as an adhesion molecule and a component of the Wnt signaling pathway. In the absence of the Wnt ligand, ${\beta}$-catenin is constantly phosphorylated, which designates it for degradation by the APC complex. This process is one of the key regulatory mechanisms of ${\beta}$-catenin. The level of ${\beta}$-catenin is also controlled by the E3 ubiquitin protein ligase SIAH-1 via a phosphorylation-independent degradation pathway. Similar to ${\beta}$-catenin, STAT3 is responsible for various cellular processes, such as survival, proliferation, and differentiation. However, little is known about how these molecules work together to regulate diverse cellular processes. In this study, we investigated the regulatory relationship between STAT3 and ${\beta}$-catenin in HEK293T cells. To our knowledge, this is the first study to report that ${\beta}$-catenin-TCF-4 transcriptional activity was suppressed by phosphorylated STAT3; furthermore, STAT3 inactivation abolished this effect and elevated activated ${\beta}$-catenin levels. STAT3 also showed a strong interaction with SIAH-1, a regulator of active ${\beta}$-catenin via degradation, which stabilized SIAH-1 and increased its interaction with ${\beta}$-catenin. These results suggest that activated STAT3 regulates active ${\beta}$-catenin protein levels via stabilization of SIAH-1 and the subsequent ubiquitin-dependent proteasomal degradation of ${\beta}$-catenin in HEK293T cells.

• Asian-Australasian Journal of Animal Sciences
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• 제25권3호
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• pp.304-310
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• 2012

The present study showed that Transforming growth factor beta 1 (TGF-${\beta}_1$) can induce apoptosis of bovine mammary epithelial cells. This apoptosis was also observed with phosphorylation of Smad2/3 within 0.5-2 h. Afterwards the signal transferred into the nucleus. Moreover, intracellular free $Ca^{2+}$ concentration was significantly elevated as well as Caspase-3 activated and DNA lysised, thereby inducing the programmed cell death. This signaling pathway of TGF-${\beta}_1$ was blocked by SB-431542 ($10^{-2}{\mu}M$) via inhibiting ALK-5 kinase activity, which thus reversed the anti-proliferation and apoptosis effect of TGF-${\beta}_1$ in mammary epithelial cells. These results indicated that TGF-${\beta}_1$ induced apoptosis of bovine mammary epithelial cells through the ALK-5-Smad2/3 pathway, which plays an important role in inhibiting survival of mammary epithelial cells. Moreover, intracellular $Ca^{2+}$ also played a critical role in TGF-${\beta}_1$-induced cell apoptosis.

• 대한수의학회지
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• 제46권2호
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• pp.149-158
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• 2006

To determine the effects of ${\beta}$-carotene on the control of mastitis in dairy cows during the dry period, 38 dairy cows (18 mastitic cows and 20 healthy cows) were administered with 5 ml of ${\beta}$-carotene (30 mg/ml) intramuscularly twice (4 week intervals). Blood samples were taken from the cows before the injection and two weeks after the second injection, respectively, and were measured for the proportion of lymphocyte subpopulations and lymphocyte proliferation responses. With ${\beta}$-carotene injection, the proportion of cells expressing BoCD2, BoCD4 and B and N cells increased in healthy cows. In the presence of mitogens, lymphocytes from healthy cows showed significantly higher proliferation responses after ${\beta}$-carotene injection than before (p < 0.05), The somatic cell counts in ${\beta}$-carotene injected group decreased from 1,001,00 cells/ml at dry off to 647,000 cell/ml and 447 cells/ml at the stage of first and second weeks after calving, respectively (p < 0,05), This study indicated that ${\beta}$-carotene as a nonspecific immunostimulator could have a definite role for the prevention of intramammary infections in dairy cows at dry period.