• Journal of Microbiology and Biotechnology
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• 제33권3호
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• pp.339-347
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• 2023

Transforming growth factor-β is a key factor in regulating adhesion formation during tendon healing. We investigated the effectiveness of SMAD family members, SMAD7 and SMAD3, in the TGF-β/Smad signaling during flexor tendon repair. Mouse flexor toe deep tendon rupture anastomosis models were made. On days 3, 7, 14, 21, and 28, the expressions of smad7 and smad3 in flexor tendon tissues were detected by RT-qPCR and western blot. Furthermore, postoperative intraperitoneal injections of SMAD7 agonists or SMAD3 antagonists were given. The degree of tendon healing was evaluated by adhesion testing and biomechanical experiments. Hematoxylin and eosin (HE) staining was used to observe the pathological changes. Immunohistochemistry was used to evaluate the expressions of collagen III, SMAD3, and SMAD7. The mRNA levels of matrix metalloproteinases, Mmp2 and Mmp9, and scleraxis (SCX) in flexor tendon tissue were detected by RT-qPCR. Smad3 expression increased and Smad7 expression decreased in flexor tendon tissue after injury. In addition, the SMAD7 agonist blocked SMAD3 phosphorylation. SMAD7 agonist and SMAD3 antagonist both improved adhesion formation during flexor tendon healing, and decreased the expressions of collagen III, Mmp9, and SCX, while increasing Mmp2 expression. This study provides a possible theoretical basis for the SMAD7-SMAD3 signal cascade during flexor tendon adhesion healing.

• 대한소아치과학회지
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• 제28권2호
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• pp.238-246
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• 2001

본 실험은 탁월한 골유도능으로 관심의 대상이 되고 있는 BMP의 세포내 신호 전달자로 알려진 Smad 1과 Smad 5가 조골세포 초기 분화표지인자인 ALP 유전자의 발현에 미치는 영향 및 그 조절기전을 알아보고자 하였다. BMP 처리 없이도 Smad에 의해 ALP가 발현되는가를 알아보기 위해 Smad 1과 Smad 5가 각각 stably transfection된 C2C12 세포를 3일간 배양후 histochemical assay를 하였고, Smad 1과 Smad 5의 expression vector와 ALP promoter vector를 transient co-transfection한 후 ALP promoter activity를 측정하였다. Smad에 의한 BMP의 효과를 알아보기 위해서 100ng/ml의 BMP-2를 처리한 군과 처리하지 않은 군으로 나누어 세포를 배양한후 ALP 유전자의 발현을 northern blot analysis로 확인 하였다. Smad가 ALP 유전자의 발현을 직접적으로 조절하는가를 알아보기 위해서는 단백질 합성억제제인 cycloheximide를 전처리하여 ALP 유전자의 발현을 northern blot analysis하였다. 이상의 실험결과 다음과 같은 결론을 얻었다. $\cdot$ Smad 1과 Smad 5가 과발현된 세포에서는 BMP 처리없이도 ALP가 발현된다. $\cdot$ Smad 1과 Smad 5가 과발현된 세포에서 BMP 처리후 ALP 발현 증가율이 대조군 보다 현저히 높게 나타나 Smad가 BMP 효과를 증가시킨다는 것을 알 수 있다. $\cdot$ Smad는 새로운 단백질의 합성을 통해 ALP 유전자를 발현시킨다.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제35권2호
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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.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2006년도 추계학술대회
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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.

• Molecules and Cells
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• 제38권1호
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• pp.20-25
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• 2015

TGF-${\beta}$ regulates pleiotropic cellular responses including cell growth, differentiation, migration, apoptosis, extracellular matrix production, and many other biological processes. Although non-Smad signaling pathways are being increasingly reported to play many roles in TGF-${\beta}$-mediated biological processes, Smads, especially receptor-regulated Smads (R-Smads), still play a central mediatory role in TGF-${\beta}$ signaling for epithelial-mesenchymal transition. Thus, the biological activities of R-Smads are tightly regulated at multiple points. Inhibitory Smad (I-Smad also called Smad7) acts as a critical endogenous negative feedback regulator of Smad-signaling pathways by inhibiting R-Smad phosphorylation and by inducing activated type I TGF-${\beta}$ receptor degradation. Roles played by Smad7 in health and disease are being increasingly reported, but the molecular mechanisms that regulate Smad7 are not well understood. In this study, we show that E3 ubiquitin ligase Itch acts as a positive regulator of TGF-${\beta}$ signaling and of subsequent EMT-related gene expression. Interestingly, the Itch-mediated positive regulation of TGF-${\beta}$ signaling was found to be dependent on Smad7 ubiquitination and its subsequent degradation. Further study revealed Itch acts as an E3 ubiquitin ligase for Smad7 polyubiquitination, and thus, that Itch is an important regulator of Smad7 activity and a positive regulator of TGF-${\beta}$ signaling and of TGF-${\beta}$-mediated biological processes. Accordingly, the study uncovers a novel regulatory mechanism whereby Smad7 is controlled by Itch.

• International Journal of Oral Biology
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• 제34권2호
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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.

• Asian Pacific Journal of Cancer Prevention
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• 제13권4호
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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.

• 한국발생생물학회지:발생과생식
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• 제11권1호
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• pp.43-47
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• 2007

Transforming growth $factor-{\beta}(TGF-{\beta})$ 신호의 매개자 역할을 하는 Smad 계열 단백질은 발생과정에 중요한 역할을 한다고 알려져 있다. Estrogen receptor(ER)와 구조적으로 유사한 estrogen receptor-related receptor(ERR)은 포유동물에서 후기 배발생기에 외배엽 형성과 관련이 되어 있는 고아핵수용체이다. 본 연구에서는 해양무척추동물의 초기발생과정과 계절번식기 동안에 Smad3와 ERR의 유전자 발현이 발생과정과 성숙에 어떠한 연관성을 갖고 있는지 알아보기 위하여, 동해안 연안에 주로 서식하는 극피동물문 둥근성게과 둥근성게(Strongylocentrotus nudus)를 재료로 하여 계절별 및 배발생 과정중에 Smad3와 $ERR{\beta}$ like 1의 mRNA 농도를 real-time PCR 방법으로 조사하였다. Smad3 mRNA는 샘플링을 시작한 2004년 2월의 생식소와 비교하면 4월부터 그 농도가 증가하기 시작하여 6월까지 증가하였으며, 산란기인 8월에 감소하였다가 10월부터 12월까지 높은 수준을 유지하였다. $ERR{\beta}$ like 1 mRNA는 6월까지 낮은 수준이었으나, 산란기인 8월에 급증한 후 다시 감소하였다. 수정란부터 초기 유생기까지 발생과정을 분석한 결과, Smad3 mRNA는 8세포기 및 16세포기에 높은 발현이 관측되었다. 한편, $ERR{\beta}$ like 1 mRNA는 포배기, 낭배기, 초기 유생기에 현저하게 높은 발현패턴을 보였다. 이상의 결과로부터 둥근성게의 산란기 및 발생배의 발생후기에 $ERR{\beta}$ like 1이 중요한 역할을 담당할 것으로 추정되며, 초기 난할시기에는 Smad3의 관련성이 시사되었다.

• BMB Reports
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• 제43권8호
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• pp.554-560
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• 2010

Smad4 is involved in cancer progression and metastasis. Using a pair of human syngeneic epithelial ovarian cancer cells with low (HO-8910) and high (HO-8910PM) metastatic abilities, we aimed to reveal the role of Smad4 in ovarian cancer metastasis in vitro. Smad4 was down-regulated in HO-8910PM cell line relative to HO-8910 by implicating Smad4 was probably a potential tumor suppressor gene for ovarian cancer. Re-expression of Smad4 decreased the migration ability and inhibited the invasion capacity of HO-8910PM, while promoted the cell adhesion capacity for HO-8910PM. The stable expression of Smad4 increased the expression of E-cadherin, reduced the expression of plasminogen activator inhibitor-1 (PAI-1) and slightly down-regulated the expression of VEGF. Smad4 suppresses human ovarian cancer cell metastasis potential through its effect on the expressions of PAI-1, E-cadherin and VEGF. Results from current work implicate Smad4 might suppress the invasion and metastasis of human ovarian tumor cells through a TGF-$\beta$/Smad-mediated pathway.

• Journal of Nutrition and Health
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• 제51권1호
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• pp.23-30
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• 2018

Purpose: Runx2 (runt-related transcription factor 2), a bone-specific transcription factor, is a key regulator of osteoblast differentiation and its expression is induced by the activation of BMP-2 signaling. This study examined whether zinc modulates BMP-2 signaling and therefore stimulates Runx2 and osteoblast differentiation gene expression. Methods: Two osteoblastic MC3T3-E1 cell lines (subclones 4 as a high osteoblast differentiation and subclone 24 as a low osteoblastic differentiation) were cultured in an osteogenic medium (OSM) as the normal control, Zn-($1{\mu}M$ Zn) or Zn+($15{\mu}M$ Zn) for 24 h. The genes and proteins for BMP-2 signaling (BMP-2, Smad-1/p-Smad-1), transcription factors (Runx2, osterix), and osteoblast differentiation marker proteins were assessed. Results: In both cell lines, BMP-2 mRAN and protein expression and extracellular BMP-2 secretion all decreased in Zn-. The expression of Smad-1 (downstream regulator of BMP-2 signaling) and p-Smad-1 (phosphorylated Smad-1) also downregulated in Zn-. Furthermore, the expression of the bone-specific transcription factors, Runx2 and osterix, decreased in Zn-, which might be due to the decreased BMP-2 expression and Smad-1 activation (p-Smad-1) by Zn-, because Runx2 and osterix both are downstream in BMP-2 signaling. Bone marker gene expression, such as alkaline phosphatase (ALP), collagen type I (COLI), osteocalcin, and osteopontin were also downregulated in Zn-. Conclusion: The results suggest that a zinc deficiency in osteoblasts suppresses the BMP-2 signaling pathway via the suppression of Smad-1 activation, and this suppressed BMP-2 signaling can cause poor osteoblast differentiation.