• 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.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.4
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• pp.1297-1303
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• 2012

Aims: Primary hepatocellular carcinoma (HCC) is a common malignancy often related to hepatitis viral infection. Smad4 is known to mediate the TGF-${\beta}$ pathway to suppress tumorigenesis. However, the function of Smad4 in HCC is still controversial. In this study we compared levels of Smad4 in HCC tissues with or without hepatitis virus infection and adjacent normal-appearing liver. Methods: Samples from HCC patients were analyzed for Smad4 protein and mRNA expression by immunohistochemistry (IHC), RT-PCR and Western blotting. Results: We found that tumor tissues expressed less Smad4 mRNA and protein than the adjacent tissues. Most HCC tumor tissues were negative for Smad4 in IHC staining, while the majority of adjacent tissues were positively stained. Interestingly, protein levels were higher in HCC tissues with viral hepatitis than those without virus infection. Suppression of expression appeared closely related to HCC, so that Smad4 appears to function as a tumor suppressor gene (TSG). Conclusion: Patients with hepatitis viral infection, at higher risk for HCC, exhibited increased Smad4 protein expression suggesting hepatitis virus may modulate Smad4 expression, which is functionally distinct from its putative role as a TSG. Smad4 expression may thus be an applicable marker for diagnosis and/or a target to develop therapeutic agents for HCC.

• IMMUNE NETWORK
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• v.12 no.3
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• pp.84-88
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• 2012

B cell-activating factor belonging to the TNF family (BAFF) is primarily expressed by macrophages and stimulates B cell proliferation, differentiation, survival, and Ig production. In this study, we explored the effect of lactoferrin (LF) on BAFF expression by murine macrophages. We determined the level of BAFF expression at the transcriptional and protein levels using RT-PCR and ELISA, respectively. LF markedly enhanced BAFF expression in mouse macrophages at both the transcriptional and protein levels. Overexpression of Smad3/4 further increased LF-induced BAFF transcription while DN-Smad3 abolished the LF-induced BAFF expression. These results demonstrate that LF can enhance BAFF expression through Smad3/4 pathway.

• IMMUNE NETWORK
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• v.11 no.4
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• pp.196-202
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• 2011

Background: B cell-activating factor belonging to the TNF family (BAFF) is primarily expressed by macrophages and dendritic cells, and stimulates B cell proliferation, differentiation, survival, and Ig production. In the present study, we explored the effect of activin A on BAFF expression by APCs. Methods: To investigate the effect of activin A on BAFF expression by mouse APCs, we measured the level of BAFF expression at the transcriptional and protein levels using RT-PCR and ELISA. Results: Activin A markedly enhanced BAFF expression in mouse macrophages and dendritic cells at both the transcriptional and protein levels. SB431542, an activin receptor-like kinase 4 (ALK4) inhibitor, completely abrogated activin A-induced BAFF transcription. Furthermore, overexpression of DN-Smad3 abolished activin-induced BAFF expression at the transcriptional and protein levels. Conclusion: These results demonstrate that activin A can enhance BAFF expression through ALK4-Smad3 pathway.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.35 no.2
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• pp.73-81
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• 2013

Purpose: Smad4 is a central mediator for transforming growth factor-${\beta}$/bone morphogenetic protein ($TGF-{\beta}/BMP$) signals, which are involved in regulating cranial neural crest cell formation, migration, proliferation, and fate determination. Accumulated evidences indicate that $TGF-{\beta}/BMP$ signaling plays key roles in the early tooth morphogenesis. However, their roles in the late tooth formation, such as cellular differentiation and matrix formation are not clearly understood. The objective of this study is to understand the roles of Smad4 in vivo during enamel and dentin formation through tissue-specific inactivation of Smad4. Methods: We generated and analyzed mice with dental epithelium-specific inactivation of the Smad4 gene (K14-Cre:$Smad4^{fl/fl}$) and dental mesenchyme-specific inactivation of Smad4 gene (Osr2Ires-Cre:$Smad4^{fl/fl}$). Results: In the tooth germs of K14-Cre:$Smad4^{fl/fl}$, ameloblast differentiation was not detectable in inner enamel epithelial cells, however, dentin-like structure was formed in dental mesenchymal cells. In the tooth germs of Osr2Ires-Cre:$Smad4^{fl/fl}$ mice, ameloblasts were normally differentiated from inner enamel epithelial cells. Interestingly, we found that bone-like structures, with cellular inclusion, were formed in the dentin region of Osr2Ires-Cre:$Smad4^{fl/fl}$ mice. Conclusion: Taken together, our study demonstrates that Smad4 plays a crucial role in regulating ameloblast and odontoblast differentiation, as well as in regulating epithelial-mesenchymal interactions during tooth development.

• Molecules and Cells
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• v.40 no.4
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• pp.291-298
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• 2017

MicroRNAs (miRNAs) are short non-coding RNAs that regulate genes posttranscriptionally. Past studies have reported that miR-210 is up-regulated in many cancers including cervical cancer, and plays a pleiotropic role in carcinogenesis. However, its role in regulating response towards anti-cancer agents has not been fully elucidated. We have previously reported that the natural compound 1'S-1'-acetoxychavicol acetate (ACA) is able to induce cytotoxicity in various cancer cells including cervical cancer cells. Hence, this study aims to investigate the mechanistic role of miR-210 in regulating response towards ACA in cervical cancer cells. In the present study, we found that ACA down-regulated miR-210 expression in cervical cancer cells, and suppression of miR-210 expression enhanced sensitivity towards ACA by inhibiting cell proliferation and promoting apoptosis. Western blot analysis showed increased expression of mothers against decapentaplegic homolog 4 (SMAD4), which was predicted as a target of miR-210 by target prediction programs, following treatment with ACA. Luciferase reporter assay confirmed that miR-210 binds to sequences in 3'UTR of SMAD4. Furthermore, decreased in SMAD4 protein expression was observed when miR-210 was overexpressed. Conversely, SMAD4 protein expression increased when miR-210 expression was suppressed. Lastly, we demonstrated that overexpression of SMAD4 augmented the anti-proliferative and apoptosis-inducing effects of ACA. Taken together, our results demonstrated that down-regulation of miR-210 conferred sensitivity towards ACA in cervical cancer cells by targeting SMAD4. These findings suggest that combination of miRNAs and natural compounds could provide new strategies in treating cervical cancer.

• Journal of Acupuncture Research
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• v.31 no.1
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• pp.7-21
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• 2014

Objectives : To observe the regenerative effects of indirect moxibustion, a traditional Korean medical treatment on skeletal muscles using mouse model of skeletal muscle adiposity. Methods : Twenty seven ICR male mice were randomly assigned into Intact control(n=3), glycerol treatment together without moxibustion(n=12), and glycerol treatment together with moxibustion (n=12) groups. Mice of glycerol treatment groups were injected with 50 ${\mu}l$ DW(distilled water) containing 50 % of glycerol into the two tibialis anterior. After injection, moxibustion was applied at 'Shenshu'($BL_{23}$) and 'Zusanli'($ST_{36}$) acupoints three times per each session, every days for twelve days(total 12 treatments). Phospho-Erk1/2, Myostatin protein levels were analyzed by western blotting and immunofluo-rescence staining techniques for tissues of the tibialis anterior muscle. Smad, phospho-Smad were analyzed by immunofluorescence staining. Results : 1. Histological analysis of sections from injected TA muscles showed that glycerol induced rapidly muscle necrosis, with a maximum at day 3. 6 days and 9 days after injection, muscle was regenerating. 2. According to western blotting and immunofluorescence staining, phospho-Erk1/2 protein signals in glycerol treatment with moxibustion group were stronger compared to Intact and glycerol treatment without moxibustion group. 3. According to western blotting and immunofluorescence staining, myostatin protein signals in glycerol treatment without moxibustion group were stronger compared to Intact and glycerol treatment with moxibustion group. 4. According to immunofluorescence staining, Smad protein signals in glycerol treatment without moxibustion group were stronger compared to Intact and glycerol treatment with moxibustion group. 5. According to immunofluorescence staining, phospho-Smad protein signals in glycerol treatment without moxibustion group were stronger compared to Intact and glycerol treatment with moxibustion group. Conclusions : These results confirm that indirect moxibustion of 'Shenshu'($BL_{23}$) and 'Zusanli'($ST_{36}$) influences muscle regeneration in mouse models of skeletal muscle adiposity. Further discussion, and the establishment of moxibustion mechanism will prompt clinical application of moxibustion.

• Molecules and Cells
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• v.45 no.6
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• pp.413-424
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• 2022

Suppressor of mothers against decapentaplegic homolog (SMAD) 4 is a pluripotent signaling mediator that regulates myriad cellular functions, including cell growth, cell division, angiogenesis, apoptosis, cell invasion, and metastasis, through transforming growth factor β (TGF-β)-dependent and -independent pathways. SMAD4 is a critical modulator in signal transduction and functions primarily as a transcription factor or cofactor. Apart from being a DNA-binding factor, the additional SMAD4 mechanisms in tumor suppression remain elusive. We previously identified methyl malonyl aciduria cobalamin deficiency B type (MMAB) as a critical SMAD4 binding protein using a proto array analysis. This study confirmed the interaction between SMAD4 and MMAB using bimolecular fluorescence complementation (BiFC) assay, proximity ligation assay (PLA), and conventional immunoprecipitation. We found that transient SMAD4 overexpression down-regulates MMAB expression via a proteasome-dependent pathway. SMAD4-MMAB interaction was independent of TGF-β signaling. Finally, we determined the effect of MMAB downregulation on cancer cells. siRNA-mediated knockdown of MMAB affected cancer cell metabolism in HeLa cells by decreasing ATP production and glucose consumption as well as inducing apoptosis. These findings suggest that SMAD4 controls cancer cell metabolism by regulating MMAB.

• The Journal of Korean Medicine
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• v.24 no.2
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• pp.1-11
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• 2003

Objectives : The aim of this study was to characterize the effect of Injinchunggan-tang on $TGF-{\beta}1-induced$ hepatic fibrosis. Methods : mRNA and protein expression levels of $TGF-{\beta}1$ in Injinchunggan-tang-treated HepG2 cells were compared to untreated cells using quantitative RT-PCR and ELISA assay, respectively. mRNA expression levels of the TGF-1 pathway genes (TR-1, TR-II, Smad2, Smad3, Smad4, and PAI-1) and fibrosis-associated genes (CTGF, fibronectin, and collagen type 1) were evaluated by quantitative RT-PCR. The effect of Injinchunggan-tang on cell proliferation of T3891 human fibroblast was evaluated using [$^3H$]thymidine incorporation assay. Results : Expression of $TGF-{\beta}1$ mRNA and protein was inhibited by Injinchunggan-tang in a dose- and time-dependent manner. Whereas $TGF-{\beta}1-mediated$ induction of PAI-1 was suppressed by Injinchunggan-tang, expression of the $TGF-{\beta}1$ pathway genes such as TR-1, TR-II, Smad2, Smad3, and Smad4 was not affected by Injinchunggan-tang treatment. Injinchunggan-tang was found to inhibit $TGF-{\beta}1-induced$ cell proliferation of T3891 human fibroblast, and also abrogated $TGF-{\beta}1-mediated$ transcriptional up-regulation of CTGF, fibronectin, and collagen type I. Conclusions : This study strongly suggests that the liver cirrhosis-suppressive activity of Injinchunggan-tang may be derived at least in part from its inhibitory effect on $TGF-{\beta}1$ functions, such as blockade of $TGF-{\beta}1$ stimulation of fibroblast cell proliferation and fibrosis-related gene expression as well as expression of $TGF-{\beta}1$ itself.

• 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.