• Molecules and Cells
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• v.43 no.4
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• pp.340-349
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• 2020

Oleoylethanolamide (OEA), a bioactive lipid in bone, is known as an endogenous ligand for G protein-coupled receptor 119 (GPR119). Here, we explored the effects of OEA on osteoclast differentiation, function, and survival. While OEA inhibits osteoclast resorptive function by disrupting actin cytoskeleton, it does not affect receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. OEA attenuates osteoclast spreading, blocks actin ring formation, and eventually impairs bone resorption. Mechanistically, OEA inhibits Rac activation in response to macrophage colony-stimulating factor (M-CSF), but not RANKL. Furthermore, the OEA-mediated cytoskeletal disorganization is abrogated by GPR119 knockdown using small hairpin RNA (shRNA), indicating that GPR119 is pivotal for osteoclast cytoskeletal organization. In addition, OEA induces apoptosis in both control and GPR119 shRNA-transduced osteoclasts, suggesting that GPR119 is not required for osteoclast apoptosis. Collectively, our findings reveal that OEA has inhibitory effects on osteoclast function and survival of mature osteoclasts via GPR119-dependent and GPR119-independent pathways, respectively.

• Toxicological Research
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• v.25 no.2
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• pp.71-78
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• 2009

We investigated the pathogenesis of liver tissue damage during the lipid peroxidation and fibrogenesis with the observation of correlations between the parameters of collagen synthesis (and deposition) and lipid peroxidation in liver fibrosis (cirrhosis) rats. Rats were randomly divided into two groups, normal and $CCl_4$-thiopental sod. intoxicated group. And the one group was treated intragastrically with the mixture of $CCl_4$-thiopental sod. 3 times per week for 3 weeks. The liver tissue and sera were used for the measurement of hydroxyproline (HYP), malonedialdehyde (MDA) and superoxide dismutase (SOD). Biochemical parameters such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total-bilirubin and blood urea nitrogen (BUN) were measured. Additionally, the expression of collagen ${\alpha}1$(III) and $\beta$-actin mRNA was observed by RTPCR. The histological change in liver tissue was also observed by Masson's trichrome and H&E staining. Correlation analysis was carried by Spearman's rho method. All biochemical parameters except total-bilirubin were significantly higher in the $CCl_4$-thiopental sod. treated group than that of the normal group (p < 0.01). In the $CCl_4$-thiopental sod. treated group, Hyp as a parameter of collagen synthesis (deposition) and MDA as a metabolite of lipid peroxidation, were significantly elevated by 1.98 and 2.11 times higher than that of the normal group (p < 0.001) respectively. The activity of SOD in the $CCl_4$-thiopental sod. treated group is decreased significantly by 44.8% (p < 0.001). And collagen ${\alpha}1$(III) mRNA was more expressed in the $CCl_4$-thiopental sod. treated group than that of the normal group. However, the expression of $\beta$-actin mRNA is showed similar in both of groups. A good correlation was observed between the content of hyp and MDA concentration (r = 0.70, n = 40) in the two groups. And the correlation between the levels of hyp and SOD (r = -0.71, n = 25) is also reliable. However, no correlation were observed between MDA concentration and SOD (r = -0.40, n = 25) in the two groups. Elevated levels of MDA in $CCl_4$-thiopental sod. treated rats indicated enhancement of lipid peroxidation, which is accompanied by a decrease in SOD activity. Moreover, we could confirm that the parameters of collagen synthesis (and deposition) is in good correlation with the metabolite of lipid peroxidation (MDA) and the lipid peroxidation antagonizing enzyme (SOD). Hence, we propose that (1) lipid peroxidation and collagen synthesis (and deposition) could be enhanced by intragastrically application of $CCl_4$-thiopental sod. during a short terms. And (2) the intoxication of $CCl_4$-thiopental sod. could be used for monitoring of lipid peroxidation and collagen synthesis (and deposition) for test of antioxidant and antifibrotic agent.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.2
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• pp.109-120
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• 2014

The epothilones are a class of microtubule inhibitors that exhibit a strong antitumor activity. UTD2 is a novel epothilone analog generated by genetic manipulation of the polyketide biosynthetic gene cluster. This study investigated the effects of UTD2 on the actin cytoskeleton and its critical regulators, and the signaling pathways which are essential for cell motility, growth and survival in MCF-7 breast cancer cells. Results showed that UTD2 inhibited the cellular functions of actin cytoskeleton, such as wound-closure, migration and invasion, as well as adhesion. Our study further demonstrated that UTD2 suppressed Rac1 GTPase activation and reduced the activity of PAK1, which is a downstream effector of Rac1, while the activity of Cdc42 was not affected. Additionally, the phosphorylation of p38 and ERK were significantly inhibited, but the phosphorylation of JNK remained the same after UTD2 treatment. Moreover, UTD2 inhibited the activity and mRNA expression of MMP-2, which plays a key role in cell motility. UTD2 also reduced the phosphorylation of Akt, which is an important signaling kinase regulating the cell survival through Rac1. Furthermore, UTD2 interrupted the synergy between Rac1 and Raf in focus formation assays. Taken together, these results indicated that UTD2 exerted multiple effects on the actin cytoskeleton and signaling pathways associated with Rac1. This study provided novel insights into the molecular mechanism of the antineoplastic and antimetastatic activities of epothilones. Our findings also suggest that the signaling pathways regulated by Rac1 may be evaluated as biomarkers for the response to therapy in clinical trials of epothilones.

• Journal of Periodontal and Implant Science
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• v.35 no.3
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• pp.623-634
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• 2005

Human periodontal ligament fibroblast(hPDLF) is very important to cure periodontal tissue because it can be diverged into various cells. This study examined the expression of MMP-1, TIMP-1, periodontal ligament specific PDLs22, Type I collagen, Fibronectin, TIMP-2, telomerase mRNA in a replicative senescence of hPDLF. The periodontal ligament tissue was obtained from periodontally healthy and non-carious human teeth extracted for orthodontic reasons at the Chosun University Hospital of Dentistry with the donors' informed consent. The hPDLF cells were cultured in a medium containing Dulbecco's modified Eagle medium(DMEM, Gibco BRL, USA) supplemented with 10% fetal bovine serum(FBS, Gibco BRL, USA) at 37C in humidified air with 5% $CO_2$. For the reverse transcription-polymerase chain reaction(RT-PCR) analysis, the total RNA of the 2, 4, 8, 16, 18, and 21 passage cells was extracted using a Trizol Reagent(Invitrogen, USA) in replicative hPDL cells. Two passage cells, i.e. young cells, served as the control, and ${\beta}-actin$ served as the internal control for RT-PCR The results of this study about cell morphology and gene expression according to aging of hPDLF using RT-PCR method are as follows: 1. The size of hPDLF was increased with aging and it was showed that the hPDLF was dying in the final passage. 2. PDLs22 mRNA was expressed in young hPDLF of the two, four, and six passage. 3. TIMP-1 mRNA was expressed in young hPDLF of the two and four passage. 4. There was a tendency that MMP-1 mRNA was weakly expressed over eighteen. 5. Type 1 collagen mRNA was expressed in almost all passages, but it was not expressed in the final passage. 6. Fibronectin mRNA was observed in all passages and it was weakly expressed in the final passage. 7. TIMP-2 and telomerase mRNA were not expressed in this study. Based on above results, it was observed that PDLs22, Type 1 collagen, Fibronectin, MMP-1. and TIMP-1 mRNA in hPDLF were expressed differently with aging. The study using the hPDLF that is collected from healthy patients and periodontitis patients needs in further study.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.2
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• pp.111-117
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• 2004

Objectives: To investigate the effect of LH receptor in folliculogenesis, we confirm the expression level of LH receptor (LH-R) mRNA in human granulosa cells (GCs) and its expression levels were analyzed by comparison to embryo developmental rate and pregnancy rate. Materials and Methods: GCs were obtained at the time of oocyte retrieval from the patients undergoing IVF-ET program. The patients were divided into two groups: Group I (n=20) is poor responder (retrieved oocyte(s)$\leq$3ea), Group II (n=80) is normal responder (retrieved oocytes>3ea). After the extraction of total RNA, semiquantitative RT-PCR was performed and the expression level of LH-R mRNA was normalized by $\beta$-actin. Statistical analysis was performed by using $X^2$ test, Student's t-test and Pearson correlation. Results: In Group II, the relative values of LH-R mRNA (0.680 vs. 0.463, p<0.005) and pregnancy rate (54.7% vs. 23.1%, p<0.05) were significantly higher than in Group I. Number of retrieved oocyte(s) was gradually increased when the expression of LH-R mRNA was increased (p<0.05). But the quality of retrieved oocyte and transferred embryo were not related with the expression of LH-R mRNA. When the pregnancy rate was compared with FSH only group and FSH combined with hMG group in the ovarian stimulation protocol, FSH combined with hMG group was significantly higher than FSH only group in Group I (37.5% vs. 0%), and the expression of LH-R mRNA was significantly higher in hMG combined group than FSH only group (p<0.05). Conclusion: Expression level of LH-R mRNA has important role in ovarian function related with the response to gonadotrophin in human folliculogenesis. Furthermore these data might provide the evidence that additional use of hMG is helpful to poor responders.

• The Journal of Internal Korean Medicine
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• v.22 no.4
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• pp.601-611
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• 2001

Objectives : We aimed to identify the dose-dependent inhibitory effects of Cheonsagunja-tang(喘四君子湯), leech(Hirudo medicinalis/水蛭) roasted with Ephedrae Herba(麻黃) on the mRNA expression of IL-6, IL-16, GM-CSF involved in the asthma model. Methods : In the study BEAS-2B cell lines, human epithelial cells were used. These cells were stimulated with tumor necrosis factor(TNF)-${\alpha}$ for artificial inflammatory expression. ${\beta}$-actin messenger RNA(mRNA) was used by internal standard. After 24 hours of the Cheonsagunja-tang, leech-treatment, total cellular RNAs were collected treating RNA zol directly on the living cells. Then the transcriptional activities of IL-6, 16 and GM-CSF were measured by RT-PCR with electrophoresis. Results: In the Cheonsagunja-tang study, the mRNA expression of IL-6 showed 30% transcriptional inhibitory effect compared to the control group in the $100{\mu}l/ml$ category(p<0.005). In the IL-16, there was 26%, 31% and 31% transcriptional inhibitory effect compared to the control groups in the $4{\mu}l/ml$, $20{\mu}l/ml$ and $100{\mu}l/ml$ categories, respectively(p<0.05). In the GM-CSF, the experimental group had 56% transcriptional inhibitory effect compared to the control group in the $100{\mu}l/ml$ category(p<0.001). In other concentrations, there was no inhibitory effect. In the leech study, the mRNA expression of IL-6 showed 37% transcriptional inhibitory effect compared to the control group in the $100{\mu}l/ml$ category(p<0.001). In the IL-16, there was 63% and 67% transcriptional inhibitory effect compared to the control groups in the $20{\mu}/ml$ and $100{\mu}/ml$ categories, respectively(p<0.001). In the GM-CSF, there was 64% and 68% transcriptional inhibitory effect compared to the control groups in the $20{\mu}l/ml$ and $100{\mu}l/ml$ categories, respectively(p<0.001). In other concentrations, there was no inhibitory effect. Conclusions : This study shows that Cheonsagunja-tang and leech roasted with Ephedrae Herba have dose-dependent inhibitory effects on the mRNA expression of IL-6, IL-16 and GM-CSF in BEAS-2B cell lines, human epithelial cells.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.14
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• pp.5815-5818
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• 2014

For an exact comparison of mRNA transcription in different samples or tissues with real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), it is crucial to select a suitable internal reference gene. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB) have been frequently considered as house-keeping genes to normalize for changes in specific gene expression. However, it has been reported that these genes are unsuitable references in some cases, because their transcription is significantly variable under particular experimental conditions and among tissues. The present study was aimed to investigate which reference genes are most suitable for the study of gastric cancer tissues using qRT-PCR. 50 pairs of gastric cancer and corresponding peritumoral tissues were obtained from patients with gastric cancer. Absolute qRT-PCR was employed to detect the expression of GAPDH, ACTB, RPII and 18sRNA in the gastric cancer samples. Comparing gastric cancer with corresponding peritumoral tissues, GAPDH, ACTB and RPII were obviously upregulated 6.49, 5.0 and 3.68 fold, respectively. Yet 18sRNA had no obvious expression change in gastric cancer tissues and the corresponding peritumoral tissues. The expression of GAPDH, ${\beta}$-actin, RPII and 18sRNA showed no obvious changes in normal gastric epithelial cells compared with gastric cancer cell lines. The carcinoembryonic antigen (CEA), a widely used clinical tumor marker, was used as a validation gene. Only when 18sRNA was used as the normalizing gene was CEA obviously elevated in gastric cancer tissues compared with peritumoral tissues. Our data show that 18sRNA is stably expressed in gastric cancer samples and corresponding peritumoral tissues. These observations confirm that there is no universal reference gene and underline the importance of specific optimization of potential reference genes for any experimental condition.

• The Journal of Advanced Prosthodontics
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• v.6 no.3
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• pp.157-164
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• 2014

PURPOSE. This study focused on in vitro cell differentiation and surface characteristics in a magnesium coated titanium surface implanted on using a plasma ion source. MATERIALS AND METHODS. 40 commercially made pure titanium discs were prepared to produce Ti oxide machined surface (M) and Mg-incorporated Ti oxide machined surface (MM). Surface properties were analyzed using a scanning electron microscopy (SEM). On each surface, alkaline phosphatase (ALP) activity, alizarin red S staining for mineralization of MC3T3-E1 cells, and quantitative analysis of osteoblastic gene expression, were evaluated. Actin ring formation assay and gene expression analysis of TRAP and GAPDH performing RT-PCR were performed to characterize osteoclast differentiation on mouse bone marrow-derived macrophages (BMMs). RESULTS. MM showed similar surface morphology and surface roughness with M, but was slightly smoother after ion implantation at the micron scale. M was more hydrophobic than MM. No significant difference between surfaces on ALP activity at 7 and 14 days were observed. Real-time PCR analyses showed similar levels of mRNA expression of the osteoblast phenotype genes; osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), and collagen 1 (Col 1) in cell grown on MM at 7, 14 and 21 days. Alizarin red S staining at 21 days showed no significant difference. BMMs differentiation increased in M and MM. Actin ring formation assay and gene expression analysis of TRAP showed osteoclast differentiation to be more active on MM. CONCLUSION. Both M and MM have a good effect on osteoblastic cell differentiation, but MM may speed the bone remodeling process by activating on osteoclast differentiation.

• Clinical and Experimental Pediatrics
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• v.55 no.10
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• pp.371-376
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• 2012

Purpose: Puromycin aminonucleoside (PAN) specifically injures podocytes, leading to foot process effacement, actin cytoskeleton disorganization, and abnormal distribution of slit diaphragm proteins. p130Cas is a docking protein connecting F-actin fibers to the glomerular basement membrane (GBM) and adapter proteins in glomerular epithelial cells (GEpCs; podocytes). We investigated the changes in the p130Cas expression level in the PAN-induced pathological changes of podocytes in vitro. Methods: We observed changes in the p130Cas expression in cultured rat GEpCs and mouse podocytes treated with various concentrations of PAN and antioxidants, including probucol, epigallocatechin gallate (EGCG), and vitamin C. The changes in the p130Cas expression level were analyzed using confocal immunofluorescence imaging, Western blotting, and polymerase chain reaction. Results: In the immunofluorescence study, p130Cas showed a diffuse cytoplasmic distribution with accumulation at distinct sites visible as short stripes and colocalized with P-cadherin. The fluorescences of the p130Cas protein were internalized and became granular by PAN administration in a dose-dependent manner, which had been restored by antioxidants, EGCG and vitamin C. PAN also decreased the protein and mRNA expression levels of p130Cas at high doses and in a longer exposed duration, which had been also reversed by antioxidants. Conclusion: These findings suggest that PAN modulates the quantitative and distributional changes of podocyte p130Cas through oxidative stress resulting in podocyte dysfunction.

• Molecules and Cells
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• v.28 no.3
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• pp.189-194
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• 2009

The modulating effect of IGF-I on the regulation of AR gene expression and activation in skeletal muscle cells remains poorly understood. In this study, the effects of IGF-I treatment on AR induction and activation in the absence of AR ligands were examined. Differentiating C2C12 cells were treated with different concentrations (0-250 ng/ml) of IGF-I or for various periods of time (0-60 min) of 250 ng/ml IGF-I. Treatment of C2C12 cells with IGF-I resulted in a dose- and time-dependent increase in total AR and phosphorylated AR (Ser 213). IGF-I treatment also led to significantly increased AR mRNA expression when compared with the control. The levels of skeletal ${\alpha}-actin$ and myogenin mRNA, known target genes of AR, were also significantly upregulated after 5 or 10 min of treatment with IGF-I. Confocal images revealed that IGF-I stimulated nuclear localization of AR in the absence of ligands. In addition, an electrophoretic mobility shift assay indicated that IGF-I stimulated the AR DNA binding activity in a time-dependent manner. The present results suggest that IGF-I stimulates the expression and activation of AR by ligand-independent mechanism in differentiating C2C12 mouse skeletal muscle cells.