• International Journal of Oral Biology
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• v.36 no.4
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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.

• Journal of the korean academy of Pediatric Dentistry
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• v.46 no.4
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• pp.409-415
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• 2019

Cleidocranial dysplasia (CCD) is an autosomal-dominant disease characterized by the delayed closure of cranial sutures, defects in clavicle formation, supernumerary teeth, and delayed tooth eruption. Defects in the Runt-related transcription factor 2 (RUNX2), a master regulator of bone formation, have been identified in CCD patients. The aim of this study was to identify the molecular genetic causes in a CCD family with delayed tooth eruption. The 23-year-old female proband and her mother underwent clinical and radiographic examinations, and all coding exons of the RUNX2 were sequenced. Mutational analysis revealed a single nucleotide deletion mutation (NM_001024630.4 : c.357delC) in exon 3 in the proband and her mother. The single C deletion would result in a frameshift in translation and introduce a premature stop codon [p.(Asn120Thrfs^*24)]. This would result in the impaired function of RUNX2 protein, which may be the cause of delayed eruption of permanent teeth in the family.

• Journal of Life Science
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• v.17 no.11
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• pp.1497-1504
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• 2007

To investigate the function and secretion of human thrombopoietin (TPO) in mammalian cells, hTPO cDNA was cloned using human liver cDNA, and recombinant hTPO (rec-hTPO) was produced in CHO cell lines. In addition, six N-linked glycosylation sites were substituted for Ala to elucidate the role of each carbohydrate chain. To analyze the biological activity, rec-hTPO protein was injected subcutaneously. Blood was withdrawn for platelet determination. The metabolic clearance rate (MCR) was also analyzed at the 1, 4, 10 and 24 hr after tail vein injection. Wild-type TPO (WT) was efficiently secreted into the medium. However, a hTPO mutant with 116 deleted nucleotides detected by PCR cloning was not secreted. The N-linked glycosylation sites had nearly the same expression quantity as rec-hTPO WT apart from mutants 3 and 4. The glycosylation site of mutant 4 appeared to be an indispensable site for hTPO secretion. Also characterized was the biological activity through an injection with rec-hTPO (10 ng) to ICR mice (7 weeks). The result of the blood analysis showed a considerable increase in the platelet number six days after He injection. To analyze the pharmacokinetics, rec-hTPO was injected into the tail vein (5 ng). The result was 200 pg/ml 1hr after this injection. Following this, it dramatically decreased and virtually disappeared 10 hours after the injection. Thus, rec-hTPO may be a treatment for thrombopenia by the production of the high active rec-hTPO. In addition, hTPO can permit the development of potent new analogues that stimulate the platelet value.

• Journal of Life Science
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• v.16 no.1
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• pp.12-16
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• 2006

Genetic alternation of Brca1 predispose of breast and ovarian cancer. Brca1 plays critical role in cell cycle regulation following DNA damage. Previous studies revealed that Brca1 plays an important role in S phase and G2/M checkpoint regulation. However, whether Brca1 involves in spindle checkpoint is unclear. In this study, the role of Brca1 in cell cycle response following nocodazole, which is a reagent that depolymerizes microtubules and activates the spindle checkpoint, has been examined using wild type $p53^{-/-}\;and\;p53^{-/-}Brca1^{-/-}$ mouse embryonic fibroblasts (MEFs). While wild type and Brca1-proficient MEFs showed an acute mitotic arrest, Brca1-deficient MEFs failed to arrest at mitotic phase in response to nocodazole treatment. In double-thymidine block and nocodazole treatment experiment, a portion of $p53^{-/-}\;Brca1^{-/-}$ MEFs were clearly by-passed nocodazole induced mitotic arrest. Consistent with this, in morphologic analysis, $p53^{-/-}\;Brca1^{-/-}$ MEFs showed growing cell morphology after nocodazole treatment. Taken together, these results suggest that Brca1 protein is an important component for normal induction of spindlecheckpoint and impairment of Brca1 function could induce dysregulation of mitotic cell cycle that ultimately results in genomic instability.

• Reproductive and Developmental Biology
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• v.33 no.3
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• pp.163-169
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• 2009

Many biological systems are regulated by an intricate set of feedback loops that oscillate with a circadian rhythm of roughly 24 h. This circadian clock mediates an increase in body temperature, heart rate, blood pressure, and cortisol secretion early in the day. Recent studies have shown changes in the amplitude of the circadian clock in the hearts and livers of streptozotocin (STZ)-treated rats. It is therefore important to examine the relationships between circadian clock genes and growth factors and their effects on diabetic phenomena in animal models as well as in human patients. In this study, we sought to determine whether diurnal variation in organ development and the regulation of metabolism, including growth and development during the juvenile period in rats, exists as a mechanism for anticipating and responding to the environment. Also, we examined the relationship between changes in growth factor expression in the liver and clock-controlled protein synthesis and turnover, which are important in cellular growth. Specifically, we assessed the expression patterns of several clock genes, including Per1, Per2, Clock, Bmal1, Cry1 and Cry2 and growth factors such as insulin-like growth factor (IGF)-1 and -2 and transforming growth factor (TGF)-${\beta}1$ in rats with STZ-induced diabetes. Growth factor and clock gene expression in the liver at 1 week post-induction was clearly increased compared to the level in control rats. In contrast, the expression patterns of the genes were similar to those observed after 5 weeks in the STZ-treated rats. The increase in gene expression is likely a compensatory change in response to the obstruction of insulin function during the initial phase of induction. However, as the period of induction was extended, the expression of the compensatory genes decreased to the control level. This is likely the result of decreased insulin secretion due to the destruction of beta cells in the pancreas by STZ.

• Journal of Life Science
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• v.18 no.4
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• pp.537-541
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• 2008

White adipose tissue is now recognized as an important endocrine organ which secretes various signal factors and proteins termed 'adipokine'. Haptoglobin (Hp), which has been known as an acute phase protein, belongs to the adipokine. However, the function of Hp in adipose tissue remains unclear. To verify the role of Hp in preadipocytes, in this study, 3T3-L1 preadipocyte cells were stably transfected with human Hp gene and Hp-overexpressing cells were made. The Hp had no effect on cell growth of preadipocytes. By RT-PCR and Western blot analysis, the Hp inhibited gene expression of IL-6 and COX-2 and enhanced HO-1 synthesis in preadipocytes. Moreover, invasion assay showed the Hp suppressed migration of monocytes to preadipocytes. These findings suggest that the Hp may inhibit an inflammatory reaction in adipose tissue by regulating the expressions of pro-inflammatory and anti-inflammatory mediators, and by repressing monocytes/macrophages infiltration.

• Korean Journal of Plant Resources
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• v.28 no.2
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• pp.168-177
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• 2015

Here we study the anti-obesity effects of by-product from soybean on mouse fed high fat diet. The body weight gain, visceral and subcutaneous adipose tissue weight, liver and epididymal adipose tissue weight in freeze-dried soybean-soaking-water (SSW) powder fed group showed lower level than those in high fat diet (HFD) group by determining with weight measuring and histological methods. Also, histological analyses of the liver and fat tissues of SSW grouped mice revealed significantly less number of lipid droplets formation and smaller size of adipocytes compared to the HFD group. Moreover, the levels of total serum cholesterol, LDL-cholesterol and the atherogenic index were decreased in the SSW groups. Especially, in SSW group, the levels of phosphorylation of two lipid oxidation enzymes, adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylasse (ACC) were elevated hence that may activate fatty acid oxidation. But AST and ALT levels were not changed in blood. By micro-CT analysis of abdomen, SSW groups significantly showed a tendency to decrease visceral and subcutaneous fats as well as fat-deposited areas compared to HFD group. Taken together, we suggest that soybean soaking water has a function in ameliorating obesity through inhibiting lipid synthesis as well as stimulating fatty acid oxidation.

• YAKHAK HOEJI
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• v.50 no.4
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• pp.272-277
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• 2006

This study was undertaken to determine whether the 9 herbal complex induces apoptosis in human breast cancer MCF-7 cells and adriamycin-resistant MCF7/adR cells. Ethanol extracts of Bojungbangamtang (BBTE) and acidic polysaccharide of Red Ginseng (GIN) induced cell death in both MCF-7 and MCF7/adR cells. Ethanol extracts of Bojungbangamtang and acidic polysaccharide of Red Ginseng also induced $G_2/M$ cell cycle arrest and increased TUNEL positive cells in MCF7/adR cells. In addition, flow cytometric analysis revealed the decreased expression of P-glycoprotein (P-gp) in ethanol extracts of Bojungbangamtang and acidic polysaccharide of Red Ginseng treated MCF7/adR cells. Similarly, decreased protein levels of P-glycoprotein and multidrug resistance associated proteins-1 were also determined by immunocytometry in ethanol extracts of Bojungbangamtang treated MCF7/adR cells. Taken together these data indicate that ethanol extracts of Bojungbangamtang and acidic polysaccharide of Red Ginseng inhibit the function of ABC transporters such as multi drug resistance associated proteins (MRPs) and P-glycoprotein as well as induce apoptosis in MCF7/adR cells. Thus, these data suggest that ethanol extracts of Bojungbangamtang and polysaccharide of Red Ginseng can be candidates for the treatment of multidrug-resistant MCF7/adR cells.

• Journal of Breast Cancer
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• v.21 no.4
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• pp.371-381
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• 2018

Purpose: Immune suppression is common in patients with advanced breast cancer but the mechanisms underlying this phenomenon have not been sufficiently studied. In this study, we aimed to identify B7 family members that were able to predict the immune status of patients, and which may serve as potential targets for the treatment of breast cancer. We also aimed to identify microRNAs that may regulate the expression of B7 family members. Methods: The Cancer Genome Atlas data from 1,092 patients with breast cancer, including gene expression, microRNA expression and survival data, were used for statistical and survival analyses. Polymerase chain reaction and Western blot were used to measure messenger RNA and protein expression, respectively. Luciferase assay was used to investigate direct microRNA target. Results: Bioinformatic analysis predicted that microRNA (miR)-93, miR-195, miR-497, and miR-340 are potential regulators of the immune evasion of breast cancer cells, and that they exert this function by targeting CD274, PDCD1LG2, and NCR3LG1. We chose CD274 for further investigations. We found that miR-195, miR-497, and CD274 expression levels were inversely correlated in MDA-MB-231 cells, and miR-195 and miR-497 expressions mimic inhibited CD274 expression in vitro. Mechanistic investigations demonstrated that miR-195 and miR-497 directly target CD274 3' untranslated region. Conclusion: Our data indicated that the level of B7 family members can predict the prognosis of breast cancer patients, and miR-195/miR-497 regulate CD274 expression in triple negative breast cancer. This regulation may further influence tumor progression and the immune tolerance mechanism in breast cancer and may be able to predict the effect of immunotherapy on patients.

• Animal Bioscience
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• v.35 no.11
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• pp.1787-1799
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• 2022

Objective: Choline deficiency, one main trigger for nonalcoholic fatty liver disease (NAFLD), is closely related to lipid metabolism disorder. Previous study in a choline-deficient model has largely focused on gene expression rather than gene structure, especially sparse are studies regarding to alternative splicing (AS). In modern life science research, primary hepatocytes culture technology facilitates such studies, which can accurately imitate liver activity in vitro and show unique superiority. Whereas limitations to traditional hepatocytes culture technology exist in terms of efficiency and operability. This study pursued an optimization culture method for duck primary hepatocytes to explore AS in choline-deficient model. Methods: We performed an optimization culture method for duck primary hepatocytes with multi-step digestion procedure from Pekin duck embryos. Subsequently a NAFLD model was constructed with choline-free medium. RNA-seq and further analysis by rMATS were performed to identify AS events alterations in choline-deficency duck primary hepatocytes. Results: The results showed E13 (embryonic day 13) to E15 is suitable to obtain hepatocytes, and the viability reached over 95% by trypan blue exclusion assay. Primary hepatocyte retained their biological function as well identified by Periodic Acid-Schiff staining method and Glucose-6-phosphate dehydrogenase activity assay, respectively. Meanwhile, genes of alb and afp and specific protein of albumin were detected to verify cultured hepatocytes. Immunofluorescence was used to evaluate purity of hepatocytes, presenting up to 90%. On this base, choline-deficient model was constructed and displayed significantly increase of intracellular triglyceride and cholesterol as reported previously. Intriguingly, our data suggested that AS events in choline-deficient model were implicated in pivotal biological processes as an aberrant transcriptional regulator, of which 16 genes were involved in lipid metabolism and highly enriched in glycerophospholipid metabolism. Conclusion: An effective and rapid protocol for obtaining duck primary hepatocytes was established, by which our findings manifested choline deficiency could induce the accumulation of lipid and result in aberrant AS events in hepatocytes, providing a novel insight into various AS in the metabolism role of choline.