Study of TGF-β/Smad3 Signal Pathway Using the Technology of Flurorescence Resonance Energy Transfer_Journal of Biomedical Engineering

Authors：

CAOWeiwei ¹ , LIUWei ¹ , WANGWeishan ² , CHENZhao ² , HERenhao ² ,  HEJianwei ³

1. Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Medical College, Shihezi University, Shihezi 832000, China;
2. Department of Orthopedics, The First Affiliated Hospital of the Medical College, Shihezi University, Shihezi 832000, China;
3. Department of Clinical Laboratory, The First Affiliated Hospital of the Medical College, Shihezi University, Shihezi 832000, China;

Corresponding author：

HEJianwei, Email: hjw5566@126.com

Keywords：

transforming growth factor-β1; Smad3; biosensor; flurorescence resonance energy transfer

DOI：

10.7507/1001-5515.20140203

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

The transforming growth factor-β1 (TGF-β1)/Smad3 signal pathway is related to mutiple physiological and pathological generation mechanism of human being. Up to date, however, the spacial and time information on the phosphorylated Smad3 is still unclear. In this study, the process of Smad3 phosphorylation was observed under the physiological state in the living cells. Firstly, the ECFP-Smad3-Citrine (Smad3 biosensor) fusion protein expression vector was constructed and identified. Then the Smad3 biosensor was transfected into 293T cells. The transfection efficiency and the expressions of fusion proteins were observed in 24 hours. Thirdly, Smad3 biosensor flurorescence resonance energy transfer (FRET) was observed with the inversion fluorescence microscope and measured by the MetaFlour FRET 4.6 software. Smad3 biosensor transfection efficiency was nearly 40% and the fusion protein was seen under the fluorescence microscope. The FRET ratio of Smad3 biosensor in living 293T cells was decreased after 10 minutes incubation with the ligand of TGF-β1. The period of decreasing CFP and enhancing Citrine signals was about 300 seconds. With the technology of FRET, the TGF-β1/Smad3 signal pathway could be real time monitored dynamically under the physiological condition in living cells.

Citation： CAOWeiwei, LIUWei, WANGWeishan, CHENZhao, HERenhao, HEJianwei. Study of TGF-β/Smad3 Signal Pathway Using the Technology of Flurorescence Resonance Energy Transfer. Journal of Biomedical Engineering, 2014, 31(5): 1080-1084. doi: 10.7507/1001-5515.20140203 Copy

1.	WOZNEY J M, ROSEN V, CELESTE A J, et al. Novel regulators of bone formation: molecular clones and activities[J]. Science, 1988, 242(4885): 1528-1534.
2.	HAYASHI H, ABDOLLAH S, QIU Y, et al. The MAD-related protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGF beta signaling[J]. Cell, 1997, 89(7): 1165-1173.
3.	SHI Y, MASSAGUÉ J. Mechanisms of TGF-β signaling from cell membrane to the nucleus[J]. Cell, 2003, 113(6):685-700.
4.	QIN B Y, LAM S S, CORREIA J J, et al. Smad3 allostery links TGF-beta receptor kinase activation to transcriptional control[J]. Genes Dev, 2002, 16(15): 1950-1963.
5.	CHEN Z, GUO K, TOH S Y, et al. Mitochondria localization and dimerization are required for CIDE-B to induce apoptosis[J]. J Biol Chem, 2000, 275(30): 22619-22622.
6.	SZILVAY G R, BLENNER M A, SHUR O, et al. A FRET-based method for probing the conformational behavior of an intrinsically disordered repeat domain from Bordetella pertussis adenylate cyclase[J]. Biochemistry, 2009, 48(47): 11273-11282.
7.	LU Y Y, CHEN T S, WANG X P, et al. Single-cell analysis of dihydroartemisinin-induced apoptosis through reactive oxygen species-mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques[J]. J Biomed Opt, 2010, 15(4): 46028.
8.	GRIESBECK O, BAIRD G S, CAMPBELL R E, et al. Reducing the environmental sensitivity of yellow fluorescent protein.mechanism and applications[J]. J Biol Chem, 2001, 276(31): 29188-29194.
9.	NOHE A, KEATING E, KNAUS P, et al. Signal transduction of bone morphogenetic protein receptors[J]. Cell Signal, 2004, 16(3): 291-299.
10.	SCHMIERER B, HILL C S. Kinetic analysis of Smad nucleocytoplasmic shuttling reveals a mechanism for transforming growth factor beta-dependent nuclear accumulation of Smads[J]. Mol Cell Biol, 2005, 25(22): 9845-9858.
11.	ITOH S, TEN DIJKE P. Negative regulation of TGF-beta receptor/Smad signal transduction [J]. Curr Opin Cell Biol, 2007,19(2): 176-184.
12.	KOHN E A, YANG Y A, DU Z J, et al. Biological responses to TGF-β in the mammary epithelium show a complex dependency on Smad3 gene dosage with important implications for tumor progression[J]. Mol Cancer Res, 2012, 10(10): 1389-1399.
13.	NAM K T, O'NEAL R, LEE Y S, et al. Gastric tumor development in Smad3-deficient mice initiates from forestomach/glandular transition zone along the lesser curvature[J]. Lab Invest, 2012, 92(6): 883-895.

1. WOZNEY J M, ROSEN V, CELESTE A J, et al. Novel regulators of bone formation: molecular clones and activities[J]. Science, 1988, 242(4885): 1528-1534.
2. HAYASHI H, ABDOLLAH S, QIU Y, et al. The MAD-related protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGF beta signaling[J]. Cell, 1997, 89(7): 1165-1173.
3. SHI Y, MASSAGUÉ J. Mechanisms of TGF-β signaling from cell membrane to the nucleus[J]. Cell, 2003, 113(6):685-700.
4. QIN B Y, LAM S S, CORREIA J J, et al. Smad3 allostery links TGF-beta receptor kinase activation to transcriptional control[J]. Genes Dev, 2002, 16(15): 1950-1963.
5. CHEN Z, GUO K, TOH S Y, et al. Mitochondria localization and dimerization are required for CIDE-B to induce apoptosis[J]. J Biol Chem, 2000, 275(30): 22619-22622.
6. SZILVAY G R, BLENNER M A, SHUR O, et al. A FRET-based method for probing the conformational behavior of an intrinsically disordered repeat domain from Bordetella pertussis adenylate cyclase[J]. Biochemistry, 2009, 48(47): 11273-11282.
7. LU Y Y, CHEN T S, WANG X P, et al. Single-cell analysis of dihydroartemisinin-induced apoptosis through reactive oxygen species-mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques[J]. J Biomed Opt, 2010, 15(4): 46028.
8. GRIESBECK O, BAIRD G S, CAMPBELL R E, et al. Reducing the environmental sensitivity of yellow fluorescent protein.mechanism and applications[J]. J Biol Chem, 2001, 276(31): 29188-29194.
9. NOHE A, KEATING E, KNAUS P, et al. Signal transduction of bone morphogenetic protein receptors[J]. Cell Signal, 2004, 16(3): 291-299.
10. SCHMIERER B, HILL C S. Kinetic analysis of Smad nucleocytoplasmic shuttling reveals a mechanism for transforming growth factor beta-dependent nuclear accumulation of Smads[J]. Mol Cell Biol, 2005, 25(22): 9845-9858.
11. ITOH S, TEN DIJKE P. Negative regulation of TGF-beta receptor/Smad signal transduction [J]. Curr Opin Cell Biol, 2007,19(2): 176-184.
12. KOHN E A, YANG Y A, DU Z J, et al. Biological responses to TGF-β in the mammary epithelium show a complex dependency on Smad3 gene dosage with important implications for tumor progression[J]. Mol Cancer Res, 2012, 10(10): 1389-1399.
13. NAM K T, O'NEAL R, LEE Y S, et al. Gastric tumor development in Smad3-deficient mice initiates from forestomach/glandular transition zone along the lesser curvature[J]. Lab Invest, 2012, 92(6): 883-895.

Previous Article
Comparison of Cell Elasticity Analysis Methods Based on Atomic Force Microscopy Indentation
Next Article
Establishment of Human Neuroblastoma SH-SY5Y Cell Line Stably Silencing Beclin1

Journal of Biomedical Engineering

Study of TGF-β/Smad3 Signal Pathway Using the Technology of Flurorescence Resonance Energy Transfer

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content