Abstract
The transforming growth factor-β (TGF-β) is a multifunctional cytokine critical for embryogenesis and tissue homeostasis. Alterations in TGF-β signaling pathway are observed in several types of malignant tumors and often related with cancer progression and metastasis. TGF-β signaling is transduced across the plasma membrane after ligand-receptor binding and consequent phosphorylation of the intracellular effectors SMAD2/3 by TGF-β receptors. Phosphorylated SMAD2/3 accumulates in the nucleus after complex formation with SMAD4 to act as transcription factors and regulate the expression of genes critically associated with cell proliferation and differentiation. Traditional methodologies used to assess TGF-β signaling pathway lack accuracy and/or show poor scalability, limiting in vitro experiments and almost excluding their use in vivo. Here, we describe a fast method to quantitate TGF-β signaling pathway activity in vitro and in vivo by using adenoviral reporters. Its implementation in vitro allows quantitating cell response to TGF-β at concentrations as low as pictograms/mL. Additionally, the use of an in vivo imaging system (IVIS) enables quantitating and monitoring TGF-β signaling pathway activity over time during cancer progression, eliminating the requirement of animal euthanasia at multiple time points for this purpose. Importantly, this protocol has been consistently used in different models and effectively led to the visualization and measurement of TGF-β activity levels. Improving the sensitivity, specificity, and scalability of methods focused on characterizing this and other molecular pathways will result in a better understanding of their biology in physiological and pathological processes.
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References
Derynck R, Budi EH (2019) Specificity, versatility, and control of TGF-β family signaling. Sci Signal 12:eaav5183
Akhurst RJ, Hata A (2012) Targeting the TGFbeta signalling pathway in disease. Nat Rev Drug Discov 11:790–811
Stenvers KL, Tursky ML, Harder KW, Kountouri N, Amatayakul-Chantler S, Grail D, Small C, Weinberg RA, Sizeland AM, Zhu HJ (2003) Heart and liver defects and reduced transforming growth factor beta2 sensitivity in transforming growth factor beta type III receptor-deficient embryos. Mol Cell Biol 23:4371–4385
Teixeira AF, Ten Dijke P, Zhu HJ (2020) On-target anti-TGF-beta therapies are not succeeding in clinical cancer treatments: what are remaining challenges? Front Cell Dev Biol 8:605
Wieser R, Wrana JL, Massagué J (1995) GS domain mutations that constitutively activate T beta R-I, the downstream signaling component in the TGF-beta receptor complex. EMBO J 14:2199–2208
Souchelnytskyi S, ten Dijke P, Miyazono K, Heldin CH (1996) Phosphorylation of Ser165 in TGF-beta type I receptor modulates TGF-beta1-induced cellular responses. EMBO J 15:6231–6240
Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin CH, Miyazono K, ten Dijke P (1997) TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4. EMBO J 16:5353–5362
Zhang Y, Feng XH, Derynck R (1998) Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-beta-induced transcription. Nature 394:909–913
Pouponnot C, Jayaraman L, Massague J (1998) Physical and functional interaction of SMADs and p300/CBP. J Biol Chem 273:22865–22868
Warmflash A, Zhang Q, Sorre B, Vonica A, Siggia ED, Brivanlou AH (2012) Dynamics of TGF-beta signaling reveal adaptive and pulsatile behaviors reflected in the nuclear localization of transcription factor Smad4. Proc Natl Acad Sci U S A 109:E1947–E1956
Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, Gauthier JM (1998) Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J 17:3091–3100
Shi Y, Wang YF, Jayaraman L, Yang H, Massagué J, Pavletich NP (1998) Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-beta signaling. Cell 94:585–594
Luwor RB, Wang B, Nheu TV, Iaria J, Tsantikos E, Hibbs ML, Sieber OM, Zhu HJ (2011) New reagents for improved in vitro and in vivo examination of TGF-beta signalling. Growth Factors 29:211–218
Chen H, Ware TMB, Iaria J, Zhu HJ (2018) Live cell imaging of the TGF- beta/Smad3 signaling pathway in vitro and in vivo using an adenovirus reporter system. J Vis Exp (137):57926. https://doi.org/10.3791/57926
Luwor RB, Baradaran B, Taylor LE, Iaria J, Nheu TV, Amiry N, Hovens CM, Wang B, Kaye AH, Zhu HJ (2013) Targeting Stat3 and Smad7 to restore TGF-beta cytostatic regulation of tumor cells in vitro and in vivo. Oncogene 32:2433–2441
Luwor RB, Hakmana D, Iaria J, Nheu TV, Simpson RJ, Zhu HJ (2015) Single live cell TGF-beta signalling imaging: breast cancer cell motility and migration is driven by sub-populations of cells with dynamic TGF-beta-Smad3 activity. Mol Cancer 14:50
Zhou F, Drabsch Y, Dekker TJ, de Vinuesa AG, Li Y, Hawinkels LJ, Sheppard KA, Goumans MJ, Luwor RB, de Vries CJ, Mesker WE, Tollenaar RA, Devilee P, Lu CX, Zhu H, Zhang L, Dijke PT (2014) Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-beta signalling. Nat Commun 5:3388
Ren J, Wang Y, Ware T, Iaria J, Ten Dijke P, Zhu HJ (2020) Reactivation of BMP signaling by suboptimal concentrations of MEK inhibitor and FK506 reduces organ-specific breast cancer metastasis. Cancer Lett 493:41–54
Acknowledgements
Research on TGF-β signaling in our laboratories was funded by the Australia’s National Health and Medical Research Council (NHMRC) and Friends of the Royal Melbourne Hospital Neurosciences Foundation (H-JZ). AFT was supported by the Melbourne Research Scholarship.
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Fonseca Teixeira, A., Iaria, J., Zhu, HJ. (2022). Fast Quantitation of TGF-β Signaling Using Adenoviral Reporter. In: Zi, Z., Liu, X. (eds) TGF-Beta Signaling. Methods in Molecular Biology, vol 2488. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2277-3_2
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DOI: https://doi.org/10.1007/978-1-0716-2277-3_2
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