Dynamic Visualization of TGF-β/SMAD3 Transcriptional Responses in Single Living Cells

Simple Summary How a single cytokine can induce a variety of cellular responses in the same cell or in different cells is a longstanding question. Transforming growth factor β (TGF-β) is a prototypical multifunctional cytokine of which biological responses are highly dependent on in a cellular context. TGF-β signals via intracellular SMAD transcription factors, and the duration and intensity of SMAD activation are key determinants for the responses that are elicited by TGF-β. To visualize the TGF-β signaling kinetics, we developed a dynamic TGF-β/SMAD3 transcriptional reporter using a quickly folded and highly unstable green florescent protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-β-induced responses in cells cultured on plastic dishes, and in living animals. This tool allows for the analysis of TGF-β signaling at a single living cell level, and allows for the discovery of dynamic TGF-β SMAD- induced transcriptional responses in multi-step biological processes. Abstract Transforming growth factor-β (TGF-β) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-β/SMAD3 signaling kinetics, we developed a dynamic TGF-β/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-β/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-β challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-β/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes.

[1]  Mauro J. Muraro,et al.  Linking the genotypes and phenotypes of cancer cells in heterogenous populations via real-time optical tagging and image analysis , 2022, Nature Biomedical Engineering.

[2]  Jing Zhang,et al.  Studying TGF-β Signaling and TGF-β-induced Epithelial-to-mesenchymal Transition in Breast Cancer Cells. , 2020, Journal of visualized experiments : JoVE.

[3]  R. Jain,et al.  A framework for advancing our understanding of cancer-associated fibroblasts , 2020, Nature Reviews Cancer.

[4]  J. van Rheenen,et al.  Fsp1-Mediated Lineage Tracing Fails to Detect the Majority of Disseminating Cells Undergoing EMT , 2019, Cell reports.

[5]  Hans Clevers,et al.  Cancer modeling meets human organoid technology , 2019, Science.

[6]  C. Hill,et al.  TGF-β family ligands exhibit distinct signalling dynamics that are driven by receptor localisation , 2019, Journal of Cell Science.

[7]  F. Naef,et al.  Quantitative relationships between SMAD dynamics and target gene activation kinetics in single live cells , 2018, bioRxiv.

[8]  Hong-Jian Zhu,et al.  Live Cell Imaging of the TGF- β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System. , 2018, Journal of visualized experiments : JoVE.

[9]  A. Loewer,et al.  Cell‐specific responses to the cytokine TGFβ are determined by variability in protein levels , 2018, Molecular systems biology.

[10]  L. Goentoro,et al.  Sensing relative signal in the Tgf-β/Smad pathway , 2017, Proceedings of the National Academy of Sciences.

[11]  Hayley E. Francies,et al.  Prospective Derivation of a Living Organoid Biobank of Colorectal Cancer Patients , 2015, Cell.

[12]  Elaine Fuchs,et al.  TGF-β Promotes Heterogeneity and Drug Resistance in Squamous Cell Carcinoma , 2015, Cell.

[13]  T. Nheu,et al.  Single live cell TGF-β signalling imaging: breast cancer cell motility and migration is driven by sub-populations of cells with dynamic TGF-β-Smad3 activity , 2015, Molecular Cancer.

[14]  M. Spector,et al.  Organoid Models of Human and Mouse Ductal Pancreatic Cancer , 2015, Cell.

[15]  Katharina F. Sonnen,et al.  Dynamic signal encoding--from cells to organisms. , 2014, Seminars in cell & developmental biology.

[16]  C. Hill,et al.  Response to comment on “Controlling long-term signaling: Receptor dynamics determine attenuation and refractory behavior of the TGF-β pathway”—Smad2/3 activity does not predict the dynamics of transcription , 2014, Science Signaling.

[17]  Aryeh Warmflash,et al.  Encoding of temporal signals by the TGF-β pathway and implications for embryonic patterning. , 2014, Developmental cell.

[18]  C. Hill,et al.  Controlling Long-Term Signaling: Receptor Dynamics Determine Attenuation and Refractory Behavior of the TGF-β Pathway , 2013, Science Signaling.

[19]  L. Saiz,et al.  Computational modelling of Smad-mediated negative feedback and crosstalk in the TGF-β superfamily network , 2013, Journal of The Royal Society Interface.

[20]  G. Lahav,et al.  Encoding and Decoding Cellular Information through Signaling Dynamics , 2013, Cell.

[21]  J. Massagué.  TGFβ signalling in context , 2012, Nature Reviews Molecular Cell Biology.

[22]  Douglas A. Chapnick,et al.  Dynamics of TGF‐β/Smad signaling , 2012, FEBS letters.

[23]  Jeff Porter,et al.  USP4 is regulated by AKT phosphorylation and directly deubiquitylates TGF-β type I receptor , 2012, Nature Cell Biology.

[24]  A. Brivanlou,et al.  Dynamics of TGF-β signaling reveal adaptive and pulsatile behaviors reflected in the nuclear localization of transcription factor Smad4 , 2012, Proceedings of the National Academy of Sciences.

[25]  Edda Klipp,et al.  Quantitative analysis of transient and sustained transforming growth factor-β signaling dynamics , 2011, Molecular systems biology.

[26]  S. Rees,et al.  Principles of early drug discovery , 2011, British journal of pharmacology.

[27]  Hans Clevers,et al.  Intestinal Crypt Homeostasis Results from Neutral Competition between Symmetrically Dividing Lgr5 Stem Cells , 2010, Cell.

[28]  M. Bertagnolli,et al.  Molecular origins of cancer: Molecular basis of colorectal cancer. , 2009, The New England journal of medicine.

[29]  Erik Sahai,et al.  Localised and reversible TGFβ signalling switches breast cancer cells from cohesive to single cell motility , 2009, Nature Cell Biology.

[30]  Xin Lu,et al.  Imaging transforming growth factor-β signaling dynamics and therapeutic response in breast cancer bone metastasis , 2009, Nature Medicine.

[31]  Margaret S. Ebert,et al.  MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells , 2007, Nature Methods.

[32]  L. Luo,et al.  A global double‐fluorescent Cre reporter mouse , 2007, Genesis.

[33]  L. Wakefield,et al.  Lentiviral reporter constructs for fluorescence tracking of the temporospatial pattern of Smad3 signaling. , 2007, BioTechniques.

[34]  P. ten Dijke,et al.  The tumor suppressor Smad4 is required for transforming growth factor beta-induced epithelial to mesenchymal transition and bone metastasis of breast cancer cells. , 2006, Cancer research.

[35]  J. Gurdon,et al.  Morphogen gradient interpretation by a regulated trafficking step during ligand-receptor transduction. , 2005, Genes & development.

[36]  J. Kleeff,et al.  Immortalization of pancreatic stellate cells as an in vitro model of pancreatic fibrosis: deactivation is induced by matrigel and N-acetylcysteine , 2005, Laboratory Investigation.

[37]  Christopher H Contag,et al.  Global Analysis of Smad2/3-Dependent TGF-β Signaling in Living Mice Reveals Prominent Tissue-Specific Responses to Injury1 , 2005, The Journal of Immunology.

[38]  Jeffrey W Pollard,et al.  Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. , 2003, The American journal of pathology.

[39]  Takeshi Norimatsu,et al.  Encoding and Decoding , 2016 .

[40]  K. Prasad,et al.  Use of short-lived green fluorescent protein for the detection of proteasome inhibition. , 2001, BioTechniques.

[41]  C. Heldin,et al.  The L45 loop in type I receptors for TGF‐β family members is a critical determinant in specifying Smad isoform activation , 1998, FEBS letters.

[42]  C. Heldin,et al.  Identification and Functional Characterization of a Smad Binding Element (SBE) in the JunB Promoter That Acts as a Transforming Growth Factor-β, Activin, and Bone Morphogenetic Protein-inducible Enhancer* , 1998, The Journal of Biological Chemistry.

[43]  Denis Vivien,et al.  Direct binding of Smad3 and Smad4 to critical TGFβ‐inducible elements in the promoter of human plasminogen activator inhibitor‐type 1 gene , 1998, The EMBO journal.

[44]  K. Kinzler,et al.  Human Smad3 and Smad4 are sequence-specific transcription activators. , 1998, Molecular cell.

[45]  K. Kinzler,et al.  Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. , 1995, Science.

[46]  M E Greenberg,et al.  The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation , 1995, Molecular and cellular biology.

[47]  D. McEachern,et al.  Sequential alteration of peanut agglutinin binding-glycoprotein expression during progression of murine mammary neoplasia. , 1992, British Journal of Cancer.

[48]  Yibin Kang.  Imaging TGFβ Signaling in Mouse Models of Cancer Metastasis. , 2016, Methods in molecular biology.

[49]  T. Terwilliger,et al.  Engineering and characterization of a superfolder green fluorescent protein , 2006, Nature Biotechnology.

[50]  Yibin Kang,et al.  A Self-Enabling TGFβ Response Coupled to Stress Signaling: Smad Engages Stress Response Factor ATF3 for Id1 Repression in Epithelial Cells.: Smad Engages Stress Response Factor ATF3 for Id1 Repression in Epithelial Cells. , 2003 .