• Journal of the korean academy of Pediatric Dentistry
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• v.28 no.2
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• pp.238-246
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• 2001

Bone morphogenetic proteins(BMPs), members of the transforming growth factor $\beta$(TGF-$\beta$) superfamily were first identified as the factors that induce ectopic bone formation in vivo, when implanted into muscular tissue. Especially BMP-2 inhibits terminal differentiation of C2C12 myoblasts and converts them into osteoblast lineage cells. In the molecular mechanism of the signal transduction of TGF-$\beta$ and related factors, intracellular signaling proteins were identified as Smad. In previous study, it has been reported that Smad 1 and Smad 5, which belong to the R-Smad family mediate BMP signaling, were involved in the induction of osteoblast differentiation in C2C12 cells. To understnad the role of Smads involved in osteogenic transdifferentiation in C2C12 cell, in present study, after we stably transfected C2C12 cells with each. Smad(Smad 1,Smad 5) expression vector, cultured for 3 days and stained for alkaline phophatase activity. ALP activity positive cells appeared in the Smad 1, Smad 5 stably transfected cell even in the abscence of BMP. After transiently co-transfected C2C12 cells with each Smad expression vector and ALP promoter, it was examined that Smad 1 and Smad 5 expression vector had increased about 2 fold ALP promoter activity in the abscence of BMP. These result suggested that both Smad 1 and Smad 5 were involved in the intracellular BMP signals which induce osteoblast differentiation in C2C12 cells. The effect of BMP on C2C12 cells with Smad 1, Smad 5 transfected were studied by using northern blot analysis. the treatment of BMP upregulated ALP mRNA level in three groups, especially upregulation of ALP was larger in Smad 1, Smad 5 transfected cell than control group. Pretreatment with cycloheximide($10{\mu}g/ml$), a protein synthesis inhibitor resulted in blocking the ALP gene expression even in BMP(100ng/ml) treated cell. These results suggested that Smad increased the level of ALP mRNA via the synthesis of a certain transcriptional regulatory protein.

• International Journal of Oral Biology
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• v.34 no.2
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• pp.115-121
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• 2009

Bone morphogenetic protein (BMP) 2 is a potent osteogenic factor. Although both Smad1/5 and mitogenactivated protein kinases (MAPKs) are activated by BMP2, the hierarchical relationship between them is unclear. In this study, we examined if BMP2-stimulated MAPK activation is regulated by Smad1/5 or vice versa. When C2C12 cells were treated with BMP2, the activation of extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun-N-terminal kinase was evident within 5 min. The knockdown of both Smad1 and Smad5 by small interfering RNA did not affect the activation of these MAPKs. In addition, neither the overexpression of Smad1 nor Smad5 induced ERK activation. When ERK activation was induced by constitutively active MEK1 expression, the protein level and activation of Smad1 increased. Furthermore, the inhibition of constitutively active BMP receptor type IB-induced ERK activation significantly suppressed Smad1 activation. These results indicate that Smad1/5 activation is not necessary for BMP2-induced MAPK activation and also that ERK positively regulates Smad1 activation.

• Proceedings of the Korean Society of Toxicology Conference
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• 2006.11a
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• pp.19-24
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• 2006

Mothers against decapentaplegic homolog 4 (SMAD4) is a tumor suppressor gene associated with gastrointestinal carcinogenesis. The aim of the present study was to characterize more precisely its role in the development and progression of human gastric carcinoma. In this study, using tissue microarray analysis of 283 gastric cancers and related lesions, we found loss of SMAD4 protein expression in the cytoplasm (36/114, 32%) and in the nucleus (46/114, 40%) of gastric cancer cells. The loss of nuclear SMAD4 expression in primary tumors correlated significantly with poor survival, and was an independent prognostic marker in multivariate analysis. We also found a substantial decrease in SMAD4 expression at both the RNA and protein level in several human gastric carcinoma cell lines. To identify the genetic and/or epigenetic mechanisms of altered SMAD4 expression in gastric carcinoma, loss of heterozygosity (LOH), promoter hypermethylation, and exon mutations were examined. We found that LOH (20/70, 29%) and promoter hypermethylation (4/73, 5%) were associated with the loss of SMAD4 expression. SMAD4 protein levels wore also affected in certain gastric carcinoma cell lines following incubation with Mc132, a proteasome inhibitor. Taken together, our results indicate that the loss of SMAD4, especially loss of nuclear SMAD4 expression, is involved in gastric cancer progression. The loss of SMAD4 in gastric carcinomas is due to several mechanisms, including LOH, hypermethylation, and proteasome degradation.

• Biomedical Science Letters
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• v.19 no.2
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• pp.124-131
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• 2013

Genome-wide association studies (GWAS) have identified a number of common variants associated with serum liver enzyme homeostasis in population. In the previous study, single nucleotide polymorphisms (SNPs) in several genes have been reported to be associated with serum liver enzyme levels in European population. We aimed to confirm whether the genetic variation of SMAD2 (SMAD family member 2) gene influence the serum liver enzyme levels in Korean population. We genotyped variants in or near SMAD2 in a population-based sample including 994 unrelated Korean adult. Here, we performed association analysis to elucidate the possible relations of genetic polymorphisms in SMAD2 gene with serum liver enzyme levels. By examining genotype data of a total of 944 subjects in 5 hospital health promotion center, we discovered the SMAD2 gene polymorphisms are associated with serum liver enzyme levels. The common and highest significant polymorphism was rs17736760 (${\beta}$=3.51, P=5.31E-07) with glutamic oxaloacetic transferase (GOT), rs17736760 (${\beta}$=5.99, P=1.25E-05) with glutamic pyruvate transaminase (GPT), and rs17736760 (${\beta}$=15.68, P=9.93E-07) with gamma glutamyl transferase (GGT) in all group. Furthermore, the SNP rs17736760 was consistently associated with GOT (${\beta}$=5.25, P=1.72E-06), GPT (${\beta}$=9.97, P=1.16E-05), GGT (${\beta}$=26.13, P=3.43E-06) in men group. Consequently, we found statistically significant SNP in SMAD2 gene that are associated with serum levels of GOT, GPT, and GGT. In addition, these results suggest that the individuals with the minor alleles of the SNP in the SMAD2 gene may be more elevated serum liver enzyme levels in the Korean population.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.5
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• pp.447-453
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• 2013

Osteoblastic activity of nectandrin A was examined in C2C12 cells. Nectandrin A enhances the BMP-induced osteoblastic differentiation and mineralization, manifested by the up-regulation of differentiation markers (alkaline phosphatase and osteogenic genes) and increased calcium contents. In C2C12 cells co-transfected with expression vector encoding Smad4 and Id1-Luc reporter, nectandrin A increased Id1 luciferase activity in a concentration-dependent manner, when compared to that in BMP-2 treated cells, indicating that Smad signaling pathway is associated with nectandrin A-enhanced osteoblastic differentiation in C2C12 cells. In addition, nectandrin A activated p38 mitogen-activated protein kinase (MAPK) in time- and concentration-dependent manners, and phosphorylated form of pSmad1/5/8 and alkaline phosphatase activity were both decreased when the cells were pretreated with SB203580, a p38 MAPK inhibitor, suggesting that p38 MAPK might be an upstream kinase for Smad signaling pathway. Taken together, nectandrin A enhances the BMP-induced osteoblastic differentiation and mineralization of C2C12 cells via activation of p38 MAPK-Smad signaling pathway, and it has a therapeutic potential for osteoporosis by promoting bone formation.

• BMB Reports
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• v.52 no.3
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• pp.202-207
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• 2019

Keloids are the most common pathological form of trauma healing, with features that seriously affect appearance and body function, are difficult to treat and have a high recurrence rate. Emerging evidence suggests that miRNAs are involved in a variety of pathological processes and play an important role in the process of fibrosis. In this study, we investigated the function and regulatory network of miR-152-5p in keloids. The miRNA miR-152-5p is frequently downregulated in keloid tissue and primary cells compared to normal skin tissue and fibroblasts. In addition, the downregulation of miR-152-5p is significantly associated with the proliferation, migration and apoptosis of keloid cells. Overexpression of miR-152-5p significantly inhibits the progression of fibrosis in keloids. Smad3 is a direct target of miR-152-5p, and knockdown of Smad3 also inhibits fibrosis progression, consistent with the overexpression of miR-152-5p. The interaction between miR-152-5p and Smad3 occurs through the Erk1/2 and Akt pathways and regulates collagen3 production. In summary, our study demonstrates that miR-152-5p/Smad3 regulatory pathways involved in fibrotic progression may be a potential therapeutic target of keloids.

• Molecules and Cells
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• v.44 no.1
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• pp.38-49
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• 2021

Airway mucus secretion is an essential innate immune response for host protection. However, overproduction and hypersecretion of mucus, mainly composed of the gel-forming MUC5AC protein, are significant risk factors for patients with asthma and chronic obstructive pulmonary disease (COPD). The transforming growth factor β (TGFβ) signaling pathway negatively regulates MUC5AC expression; however, the underlying molecular mechanism is not fully understood. Here, we showed that TGFβ significantly reduces the expression of MUC5AC mRNA and its protein in NCI-H292 cells, a human mucoepidermoid carcinoma cell line. This reduced MUC5AC expression was restored by a TGFβ receptor inhibitor (SB431542), but not by the inhibition of NF-κB (BAY11-7082 or Triptolide) or PI3K (LY294002) activities. TGFβ-activated Smad3 dose-dependently bound to MUC5AC promoter. Notably, TGFβ-activated Smad3 recruited HDAC2 and facilitated nuclear translocation of HDAC2, thereby inducing the deacetylation of NF-κB at K310, which is essential for a reduction in NF-κB transcriptional activity. Both TGFβ-induced nuclear translocation of Smad3/HDAC2 and deacetylation of NF-κB at K310 were suppressed by a Smad3 inhibitor (SIS3). These results suggest that the TGFβ-activated Smad3/HDAC2 complex is an essential negative regulator for MUC5AC expression and an epigenetic regulator for NF-κB acetylation. Therefore, these results collectively suggest that modulation of the TGFβ1/Smad3/HDAC2/NF-κB pathway axis can be a promising way to improve lung function as a treatment strategy for asthma and COPD.

• Herbal Formula Science
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• v.26 no.3
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• pp.183-193
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• 2018

Objectives : In Traditional Korean Medicine, Epimedium koreanum Nakai has diverse pharmacological activities to treat impotence, forgetfulness, cataract and exophthalmos. Present study investigated anti-fibrogenic effects of E. koreanum water extract (EKE) in hepatic stellate cells (HSCs). Methods : To study anti-fibrogenic effects of EKE, LX-2 cells, a human immortalized HSCs, were pre-treated with $3-300{\mu}g/mL$ of EKE, and then subsequently exposed to 5 ng/mL of transforming growth $factor-{\beta}1$ ($TGF-{\beta}1$). Expression level of ${\alpha}-smooth$ muscle actin was determined by immunoblot analysis. Phosphorylation of Smad, transactivation of Smad, and expression of plasminogen activator inhibitor-1 (PAI-1) were monitored to investigate the effect of EKE on $TGF-{\beta}1-mediated$ signaling pathway. Results : Up to $100{\mu}g/mL$, EKE did not show any cytotoxicity on LX-2 cells. Pre-treatment of EKE ($100{\mu}g/mL$) significantly inhibited ${\alpha}-smooth$ muscle actin expression induced by $TGF-{\beta}1$. In addition, EKE significantly decreased Smad2 and Smad3 phosphorylations, Smad binding element-driven luciferase activity and PAI-1 expression by $TGF-{\beta}1$. Of three flavonoid compounds found in EKE, only quercertin ($30{\mu}M$) attenuated $TGF-{\beta}1-mediated$ PAI-1 expression. Conclusion : These results suggest that EKE has an ability to suppress fibrogenic process in HSCs via inhibition of $TGF-{\beta}1/Smad$ signaling pathway.

• Journal of Life Science
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• v.18 no.4
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• pp.461-466
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• 2008

Transforming growth $factor-{\beta}$ ($TGF-{\beta}$)-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. Gadd45b has been known to participate in $TGF-{\beta}-induced$ apoptosis by the activation of p38 kinase. In this report, we show that Gadd45b is an immediate-early response gene for $TGF-{\beta}$ during apoptosis in EpH4 cells. To elucidate the molecular mechanism of $TGF-{\beta}-induced$ Gadd45b gene expression, we cloned the 5'-flanking region of the mouse Gadd45b gene. When transfected into EpH4 cells, this 5'-flanking region conferred promoter activity and inducibility by $TGF-{\beta}$. Deletion analyses demonstrated that the minimal promoter activity was detected in the proximal region 220 bp upstream of the transcription initiation site. We also found that the proximal Gadd45b promoter is activated by $TGF-{\beta}$ through the action of Smad2, Smad3, and Smad4. Finally, we show that the expression of Gadd45b gene by $TGF-{\beta}$ is suppressed in EpRas cells in which $TGF-{\beta}$ could not induce apoptosis, suggesting that Gadd45b may be a crucial target for $TGF-{\beta}-induced$ apoptosis in EpH4 cells.

• Journal of dental hygiene science
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• v.10 no.5
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• pp.395-401
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• 2010

Over expression of TGF-${\beta}1$ revealed the same phenotype as NFI-C deficient mouse. It has been reported that NFI-C deficient mice demonstrated abnormal odontoblast differentiation and aberrant dentin formation during root development. In the present study, in order to investigate the histological differences between wild type (WT) mouse and NFI-C deficient mouse, we compared morphological characteristics and smad4 expression between those mice. Hematoxyline-eosin (H-E) staining was used to investigate morphological changes and immunohistochemistry was also performed to observe the Smad4 expression pattern. In H-E staining, incisor of NFI-C deficient mouse showed an open area in the lingual root, irregular odontoblasts and osteodentin. Also, NFI-C deficient mouse showed short root and osteodentin in molar. In addition, Smad4 protein was strongly expressed in NFI-C deficient mouse compared with wild type. These findings suggest that NFI-C deficiency affects odontoblast differentiation and result in the formation of abnormal roots. Therefore, balancing between NFI-C and TGF-${\beta}$ signaling including Smad4 is important for the regulation of normal odontoblast differentiation and dentin formation.