1.Relationship between expression of Smad and ventricular remodeling after myocardial infarction in rats.
Lan-feng WANG ; Lei ZHANG ; Rui-ying ZHANG ; Shao-jun LI
Chinese Journal of Cardiology 2005;33(10):932-935
OBJECTIVETo investigate the relationship between expression of Smad3, Smad7 and ventricular remodeling in rats after myocardial infarction.
METHODSMyocardial infarction was induced by left anterior descending coronary artery ligation in rats (n = 11) and sham-operated rats were used as control (n = 10). The rats were sacrificed 8 weeks later. Heart weight/body weight (HW/BW), mean blood pressure, left ventricular end diastolic pressure (LVEDP), collagen content in the un-infarcted area were examined. The mRNA levels of transforming growth factor (TGF)beta(1), Smad 3, Smad7 were determined by RT-PCR.
RESULTCompared with controls, the level of HW/BW, LVEDP and collagen content were significant increased. The mRNA expression of TGFbeta(1) and Smad3 was significantly increased in areas of myocardial infarction, border of the infarction, interventricular septum and right ventricle. The expression of Smad7 mRNA in these areas was decreased.
CONCLUSIONThese results indicated that TGFbeta(1)-Smads signaling was correlated to the ventricular remodeling after myocardial infarction. Smad3 might promote the process while Smad7 inhibit the process.
Animals ; Male ; Myocardial Infarction ; metabolism ; physiopathology ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Smad3 Protein ; genetics ; metabolism ; Smad7 Protein ; genetics ; metabolism ; Transforming Growth Factor beta ; metabolism ; Ventricular Remodeling
2.Expression of TGF-beta1/Smad protein in rat liver fibrosis model and the role of IFN-gamma.
Xiao-Qing FU ; Shou-Rong LIU ; Jian-Chun GUO ; Jian-Feng BAO
Chinese Journal of Experimental and Clinical Virology 2013;27(5):340-343
OBJECTIVETo study the impact of IFN-gamma on liver fibrosis and its possible mechanism. Thirty healthy male SD rats were randomly divided into two groups: fibrosis model group, IFN-gamma treatment group. Experimental liver fibrosis was induced by subcutaneous injection of CCl4. After 12-week-treatment, serum hyalurnic acid and TGF-beta1 was examined, histopathological changes and degrees of fibrosis were observed by optical microscopy. Meanwhile, the expression of TGF-beta1, TbetaR- I and Smad2/3 proteins was detected by immunohistochemistry and quantified by using computerized image analysis.
RESULTS(1) Pathological observation of hepatic specimens: histological examination showed that there were significant difference between normal group and fibrosis model group by comparing with the degrees of inflammation and fibrosis (P < 0.05). And the difference between fibrosis model group and IFN-gamma treatment group was significant (P < 0.05). (2) Changes of the hepatic fibrosis index (serum HA and TGF-beta1): the levels of serum HA, TGF-beta1 in fibrosis model group were higher than IFN-gamma treatment groups (P < 0.05). (3) Changes of gene protein levels about TGF-beta1/Smad: the expressions of TGF-beta1, TbetaR- I and Smad2/3 in rat hepatic tissue were detected with immunohistochemistry techniques. The expressions of the three items in model group were higher than normal group (P < 0.01). The difference between model group and IFN-gamma treatment group was significant (P < 0.05);
CONCLUSIONIFN-gamma treatment group had significant results on treating experimental hepatic fibrosis. By the way of inhibiting expressions of TGF-beta1, TbetaR- I, Smad2/3, IFN-gamma treatment group exerted its anti-fibrosis effect.
Animals ; Disease Models, Animal ; Humans ; Interferon-gamma ; therapeutic use ; Liver ; drug effects ; metabolism ; Liver Cirrhosis ; drug therapy ; genetics ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Smad2 Protein ; genetics ; metabolism ; Smad3 Protein ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism
4.Construction and activity evaluation of pSUPER RNAi system that inhibits Smad3.
Dong-shan ZHANG ; Fu-you LIU ; You-ming PENG ; Guan-zhong XIONG ; Xiang-ping CHAI
Journal of Central South University(Medical Sciences) 2007;32(6):1042-1046
OBJECTIVE:
To construct the expressing vector of siRNA which can inhibit the Smad3 activity.
METHODS:
Sixty-four bases of 2 pair oligos for hairp in RNA expression which targeted Smad3 gene were chemically synthesized and annealed. pSUPER vector was linearized with BgL II and Hin d III treated with alkaline phosphatase (CIP). Anneled oligos were inserted into the downstream of the treated pSUPER's pol III H1 promoter to construct RNAi plasmid (pSUPER Smad3). Oligos with a scrambled sequence were used as a negative control. pSUPER Smad3 was transfected into human renal tubular epithelial cells (HKC).
RESULTS:
Recombinant pSUPER Smad3 vector was identified by the digestion with Eco R I and Hin d III, and confirmed by the sequencing analysis with T3 primer. Sixty-four bases had been inserted into the expected site. Furthermore, the insertion sequence was exactly corrected. The activity evaluation indicated that mRNA and protein of Smad3 but not Smad2 were inhibited by pSUPER Smad3 in HKC.
CONCLUSION
The pSUPER Smad3 system has been constructed successfully, and has high inhibition and specificity in vitro.
Epithelial Cells
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metabolism
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Humans
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Kidney Tubules
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cytology
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Plasmids
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RNA Interference
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RNA, Messenger
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genetics
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RNA, Small Interfering
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Smad3 Protein
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antagonists & inhibitors
;
genetics
;
Transfection
5.AXIN1-related CSRNP1 mRNA expression and its transcriptional regulation in TGF-β1-induced tumor cells.
Fan DENG ; Songyu LI ; Wanfu XU ; Zhipeng ZOU ; Zhiyong KE ; Fangyin ZENG
Journal of Southern Medical University 2013;33(8):1122-1126
OBJECTIVETo investigate AXIN1-related CSRNP1 gene expression and the mechanism of its transcriptional regulation in TGF-β1-induced tumor cells.
METHODSHuman lung carcinoma A549 cells or human prostate cancer PC3 cells were treated with TGF-β1 at different doses (0, 20, 40, and 80 ng/ml) or at 20 ng/ml for 0, 8, 12, or 24 h, and the dose and time effect of TGF-β1 on CSRNP1 mRNA expression in the tumor cells were evaluated with real-time RT-PCR. A549 cells were also treated with TGF-β1 and cycloheximide to clarify whether CSRNP1 expression induced by TGF-β1 required de novo protein synthesis. A549 cells transfected with pcDNA3.1, flag-SMAD3, or flag-SMAD3-mu, after serum starvation, were treated with or without TGF-β1 (20 ng/mL) for 24 h, and the overexpression of wild-type SMAD3 and dominant negative SMAD3-mu mutant were confirmed by Western blotting. The effect of SMAD3 or SMAD3-mu overexpression on CSRNP1 mRNA expression was also measured by real-time RT-PCR.
RESULTSIn both A549 and PC3 cells, TGF-β1 dose- and time-dependently stimulated CSRNP1 expression, which required de novo protein synthesis in A549 cells. Overexpression of wild-type SMAD3 significantly increased the expression of CSRNP1 mRNA induced by TGF-β1, while overexpression of dominant negative SMAD3 mutant remarkably reduced CSRNP1 mRNA expression in response to TGF-β1 in A549 cells.
CONCLUSIONTGF-β1 may contribute to CSRNP1 expression through SMAD3 activation and downstream signaling in tumor cells.
Apoptosis Regulatory Proteins ; genetics ; metabolism ; Axin Protein ; genetics ; metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; RNA, Messenger ; genetics ; Signal Transduction ; Smad3 Protein ; genetics ; metabolism ; Transfection ; Transforming Growth Factor beta1 ; pharmacology
6.Influence of nourishing yin and tonifying yang sequential therapy combined with Western medicine on TGF-β1/Smads signaling pathway in anovulatory infertility rats with diminished ovarian reserve.
Yuying SUN ; Shuping CHEN ; Yong TAN
Journal of Central South University(Medical Sciences) 2018;43(10):1068-1074
To explore the influence for combination of nourishing yin and tonifying yang sequential therapy (NYTYST) with Western medicine in treating anovulatory infertility rats with diminished ovarian reserve (DOR) based on TGF-β1/Smads signaling pathway.
Methods: A total of 40 female rats were randomly divided into 5 groups, a normal control group, a model group, a Western medicine group, a NYTYST group and a combination group (n=8 in each group). The DOR model was established through orally taking tripterygium pill for continuous 2 weeks. The normal control group and the model group were treated with saline for 10 days. The Western medicine group was treated with hormone replacement therapy (HRT) and ovarian stimulation. The NYTYST group was treated with nourishing yin herbs in proestrus and tonifying yang herbs in late estrus and the combination group was treated with Chinese herb and Western drugs for 10 days. HE staining was used to observe histopathologic changes in ovary. Expression levels of transforming growth factor β1 receptor (TGF-β1R) in rats ovarian were detected by immunohistochemistry. Expression levels of Smad2, Smad3 and Smad7 protein in rat ovarian were detected by Western blot.
Results: Compared with the control group, the numbers of developing follicles, mature follicles and corpus luteum were decreased , while atrefic follicles were increased significantly in the model group (P<0.01); the levels of TGF-β1R, Smad2 and Smad3 were decreased significantly, while Smad7 was increased significantly (P<0.01). Compared with the model group, the numbers of developing follicles, mature follicles and corpus luteum, Smad2 and Smad3 expression were increased, while atrefic follicles and Smad7 were decreased significantly in the treatment group (P<0.05 or P<0.01). The numbers of developing follicles and corpus luteum in the combination group was superior to the Western medicine group (P<0.05). Compared with the Western medicine group, the levels of TGF-β1R, Smad2 and Smad3 were increased significantly, while Smad7 was decreased significantly in the combination group (P<0.05 or P<0.01).
Conclusion: NYTYST combined with Western medicine can improve the function of ovaries reserve by up-regulation of TGF-β1R, Smad2 and Smad3 while down-regulation of Smad7 in DOR rats.
Animals
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Drugs, Chinese Herbal
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therapeutic use
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Female
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Gene Expression Regulation
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drug effects
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Infertility
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therapy
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Medicine, Chinese Traditional
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Ovarian Reserve
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drug effects
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Rats
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Signal Transduction
;
drug effects
;
Smad2 Protein
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genetics
;
metabolism
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Smad3 Protein
;
genetics
;
metabolism
;
Transforming Growth Factor beta1
;
genetics
;
metabolism
7.The effects of the Smad3-knockout on the hematopoiesis of mouse.
Ling ZHANG ; Zhao SUN ; Ai-Ling SHEN ; Li MA ; Xue-Ying JIANG ; Guan-Jie MA ; Xiao YANG ; Chun-Hua ZHAO
Chinese Journal of Biotechnology 2003;19(4):428-432
The effects of the Smad3- knockout on the hematopoiesis of mouse were investigated in this work. Five pairs of wild type and Smad3- null mice were studied. White blood cell(WBC), red blood cell(RBC) and platelet (PLT) counting of peripheral blood cells were performed with blood obtained from tails. And white blood cells were classified by their morphology. Bone marrow nucleated cells (BMNCs) were counted and classified. The CFU-GM, BFU-E, CFU-GEMM yields were measured in each pair of mice. CFU-S yield of each mouse was measured by injecting bone marrow cells into lethally irradiated 8-10 weeks old wild type female mice. And the pathomorphism of their bone marrows, spleens and livers were observed. As a result, WBC and PLT of Smad3- null mice were significantly higher than those in wild type mice. Smad3- null mice had much more proportion of granulocytes in classification. There wasn't any difference in RBC counting and BFU-E measurement. The yield of CFU-GM increased, while the yields of CFU-GEMM and CFU-S markedly reduced. Bone marrows are actively proliferative, with granulocytosis. The granulocyte/erythrocyte ratio increased. There were no obviously alterative in spleen and liver. Thus Smad3- knockout results in a decreased number of stem and progenitor cells. Moreover hematopoietic differentiation is abnormal with a tendency to forming more granulocytes and platelets. The effect of Smad3 on hematopoiesis is correlative to that of TGF-beta.
Animals
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Bone Marrow Cells
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cytology
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metabolism
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Cell Differentiation
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Erythrocytes
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cytology
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metabolism
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Erythroid Precursor Cells
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cytology
;
metabolism
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Female
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Granulocyte-Macrophage Progenitor Cells
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cytology
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metabolism
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Granulocytes
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cytology
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metabolism
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Hematopoiesis
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genetics
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Mice
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Mice, Knockout
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Myeloid Progenitor Cells
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cytology
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metabolism
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Smad3 Protein
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genetics
8.Effect of dihydrotestosterone on the transcriptions and expressions of Smad3 and Smad4 in LNCaP cell line.
Run-guo GU ; Chun-wen ZHOU ; Qing-zheng MA
National Journal of Andrology 2006;12(3):211-214
OBJECTIVETo investigate the effect of dihydrotestosterone (DHT) on the gene transcriptions and expressions of Smad3 and Smad4 in androgen dependent prostate cancer cell line LNCaP, and whether this effect can be suppressed by the androgen receptor inhibitor flutamide.
METHODSThe androgen dependent prostate cancer cell line LNCaP was cultured in RPMI 1640 medium and treated with different concentrations of DHT(2, 10, 50 nmol/L) and flutamide (100 nmol/L). Quantitative reverse transcription PCR (RT-PCR) was used to detect the mRNAs of Smad3 and Smad4. The expressions of Smad3 and Smad4 protein were detected by Western blot assay.
RESULTSCompared with the control group without any DHT or flutamide, higher concentration(10, 50 nmol/L) of DHT enhanced the transcription of Smad3 mRNA (P <0.05). Serial concentrations of DHT increased the expression of Smad3 protein(P < 0.05). Flutamide inhibited the up-regulation of both Smad3 mRNA transcription and expression significantly (P <0.05). 10 nmol/L DHT significantly suppressed the transcription of Smad4 (P <0.05). There was considerable suppressions of Smad4 expression at the presence of DHT in different concentrations (P < 0.05). And the degree of this suppression was more significant than that of DHT on Smad4 mRNA transcription. Flutamide inhibited the suppressive effects of DHT on both Smad4 mRNA transcription and expression.
CONCLUSIONDHT can enhance the transcription and expression of Smad3, while it decreases the transcription and expression of Smad4 in LNCaP cell line. There is a possible crosstalk between the AR signal and TGF-beta signal passways at the level of Smads.
Androgens ; physiology ; Cell Line, Tumor ; Dihydrotestosterone ; pharmacology ; Flutamide ; pharmacology ; Humans ; Male ; Neoplasms, Hormone-Dependent ; metabolism ; Prostatic Neoplasms ; metabolism ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; Smad3 Protein ; biosynthesis ; genetics ; Smad4 Protein ; biosynthesis ; genetics ; Transcription, Genetic
9.Change of gene expression of transforming growth factor-beta1, Smad 2 and Smad 3 in hypertrophic scars skins.
Wei CHEN ; Xiaobing FU ; Tongzhu SUN ; Xiaoqing SUN ; Zhili ZHAO ; Zhiyong SHENG
Chinese Journal of Surgery 2002;40(1):17-19
OBJECTIVETo explore the regulatory mechanisms of transforming growth factor-beta1 (TGF-beta1) and two transcriptional factors Smad 2, 3 on hypertrophic scar formation and fetal scarless healing.
METHODSThirty-two cases were detected to compare the gene expression of TGF-beta1, Smad 2 and Smad 3 with RT-PCR. Among those cases, there were 8 cases of hypertrophic scars, 8 cases of control skins, 8 cases of fetal skins and 8 cases of adult skins.
RESULTSTGF-beta1, Smad 2 and Smad 3 gene expression could all be detected in hypertrophic scars, fetal and adult skins. Among 8 groups examinated in this experiment (each group comprised a hypertrophic scar and its corresponding normal skin), there were 5, 8 and 5 groups in which TGF-beta1, Smad 2 and Smad 3 gene expression were higher in hypertrophic scars than in normal skins respectively. The fetal skins showed significantly lower level of TGF-beta1 and Smad 3 gene expression compared with adult skins (t = 2.204, P < 0.05 and t = 4.269, P < 0.01 respectively), while mRNA contents of Smad 2 were obviously higher in fetal skins than in adult skins (t = 6.685, P < 0.01).
CONCLUSIONTGF-beta1 and its downstream signal molecules Smad 2, Smad 3 might be involved in hypertrophic scar formation. Higher gene expression of TGF-beta1, Smad 2 and Smad 3 in hypertrophic scars might lead to stimulating extracellular matrix deposition, inducing fibroblast proliferation and accelerating fibrogenesis. Lower mRNA contents of TGF-beta1 and Smad 3 in fetal skins compared with adult skins might be associated with fetal scarless healing.
Cicatrix, Hypertrophic ; metabolism ; DNA-Binding Proteins ; genetics ; Gene Expression Regulation ; Reverse Transcriptase Polymerase Chain Reaction ; Skin ; metabolism ; Smad2 Protein ; Smad3 Protein ; Trans-Activators ; genetics ; Transforming Growth Factor beta ; genetics ; Transforming Growth Factor beta1
10.TGF-beta receptor mediated telomerase inhibition, telomere shortening and breast cancer cell senescence.
Lucy CASSAR ; Craig NICHOLLS ; Alex R PINTO ; Ruping CHEN ; Lihui WANG ; He LI ; Jun-Ping LIU
Protein & Cell 2017;8(1):39-54
Human telomerase reverse transcriptase (hTERT) plays a central role in telomere lengthening for continuous cell proliferation, but it remains unclear how extracellular cues regulate telomerase lengthening of telomeres. Here we report that the cytokine bone morphogenetic protein-7 (BMP7) induces the hTERT gene repression in a BMPRII receptor- and Smad3-dependent manner in human breast cancer cells. Chonic exposure of human breast cancer cells to BMP7 results in short telomeres, cell senescence and apoptosis. Mutation of the BMPRII receptor, but not TGFbRII, ACTRIIA or ACTRIIB receptor, inhibits BMP7-induced repression of the hTERT gene promoter activity, leading to increased telomerase activity, lengthened telomeres and continued cell proliferation. Expression of hTERT prevents BMP7-induced breast cancer cell senescence and apoptosis. Thus, our data suggest that BMP7 induces breast cancer cell aging by a mechanism involving BMPRII receptor- and Smad3-mediated repression of the hTERT gene.
Actin-Related Protein 2
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genetics
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metabolism
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Activin Receptors, Type II
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genetics
;
metabolism
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Bone Morphogenetic Protein 7
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genetics
;
metabolism
;
Bone Morphogenetic Protein Receptors, Type II
;
genetics
;
metabolism
;
Breast Neoplasms
;
genetics
;
metabolism
;
Cellular Senescence
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Female
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HeLa Cells
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Humans
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MCF-7 Cells
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Neoplasm Proteins
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genetics
;
metabolism
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Protein-Serine-Threonine Kinases
;
genetics
;
metabolism
;
Receptor, Transforming Growth Factor-beta Type II
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Receptors, Transforming Growth Factor beta
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genetics
;
metabolism
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Smad3 Protein
;
genetics
;
metabolism
;
Telomerase
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genetics
;
metabolism
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Telomere Homeostasis