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      Quantitative analysis of transient and sustained transforming growth factor-β signaling dynamics

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          Abstract

          Mathematical modeling and experimental analyses reveal that TGF-β ligand depletion has an important role in converting short-term graded signaling responses to long-term switch-like responses.

          Abstract

          • Cells respond in real time to the absolute number of TGF-β molecules in their environment.

          • A single pulse of TGF-β stimulation results in transient SMAD activation whereas repeated short pulses of stimulation result in sustained SMAD activation.

          • Ligand-induced short-term TGF-β/SMAD signaling activation is graded while long-term signaling response is switch-like or ultrasensitive.

          • TGF-β ligand depletion is a major cause of conversion from graded short-term responses to ultrasensitive long-term responses.

          Abstract

          The transforming growth factor-β (TGF-β) pathway is a prominent signaling pathway that regulates diverse aspects of cellular homeostasis, including proliferation, differentiation, migration, and death ( Massague, 1998). Remarkably, the pleiotropic biological effects of TGF-β are mediated by a relatively simple signaling module ( Clarke and Liu, 2008). An interesting question is how such an apparently straightforward and simple cascade can generate a wide array of biological responses depending on the cellular context.

          Members of the TGF-β superfamily are frequently used as morphogens in early embryo development ( Green, 2002). The best-studied examples include Dpp in Drosophila and Activin in Xenopus ( Gurdon and Bourillot, 2001; Lander, 2007). In the developmental context, cells can respond to a graded ligand concentration and produce discrete biological responses (e.g., transcription of certain genes, proliferation, or differentiation; Green, 2002). To convert continuous morphogen stimulation into discrete responses, mechanisms must exist to provide a threshold for the cellular response. How variable TGF-β ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remains poorly understood.

          We have previously reported that ligand molecules per cell is the input variable to which the cells respond, and ligand number per cell is the best predictor of signaling responses ( Zi and Klipp, 2007a; Clarke et al, 2009). Here, we developed an improved mathematical model to predict TGF-β signaling responses by calibrating the model with various experimental data sets from different TGF-β stimulations. Using a combined experimental and mathematical modeling approach, we showed that TGF-β pulse stimulation results in transient activation of the pathway while repeated short pulses at short time intervals lead to a sustained activation similar to persistent ligand exposure.

          We next investigate the system response to variable doses of TGF-β in HaCaT cells. Our mathematical model predicts that the short-term Smad2 phosphorylation (after 45 min of TGF-β stimulation) is a graded response, while long-term Smad2 activation (after 24 h of TGF-β stimulation) is a switch-like response ( Figure 5A and B). As shown in Figure 5A–D, both short- and long-term Smad2 phosphorylation can be saturated but doses of TGF-β that cause maximum response are quite different. Additionally, the shapes of response curves were different. The short-term Smad2 activation was a graded (Michaelis–Menten-like) response with a very low apparent Hill coefficient of about 0.8 ( Figure 5A and C) while the long-term Smad2 activation (P-Smad2 at 24 h) yielded a switch-like response with an apparent Hill coefficient of about 4.5 ( Figure 5B and D). Thus, the Smad2 response is initially graded and sharpens over time to become ultrasensitive. To address whether TGF-β-inducible gene expression responses are graded or switch-like in the short and long term, we measured mRNA levels of Smad7, an early responsive gene of TGF-β and protein levels of p21 and PAI-1 whose inductions are delayed and late, respectively. The experimental data show that Smad7 induction exhibits a graded response with corresponding Hill coefficients of about 1.3 ( Figure 5E), which is consistent with the graded P-Smad2 response at 45 min ( Figure 5A and C). PAI-1 induction in response to variable doses of TGF-β for 24 h is highly ultrasensitive with an apparent Hill coefficient of ∼5.3. Compared with Smad7 and PAI-1, p21 induction is only modest ultrasensitive ( n Hill≈2) ( Figure 5G). These results suggest short-term gene induction by TGF-β appears to be graded while long-term targets are more switch-like. Finally, we measured the growth inhibitory response of HaCaT cells to variable doses of TGF-β. The level of BrdU incorporation is also ultrasensitive with an apparent Hill coefficient of about 4.3 ( Figure 5H). Therefore, the long-term TGF-β growth inhibitory response also shows a switch-like behavior. Finally, we show that TGF-β depletion affects long-term Smad phosphorylation and switch-like response of TGF-β signaling system. These findings shed new light on how continuous ligand doses are translated into discontinuous cell fate decisions in biological systems.

          In summary, we have shown that the dose and time course of TGF-β stimulation have profound effects on Smad signaling dynamics. The rate of ligand depletion controls the duration of Smad2 phosphorylation. Cells can respond to a short pulse of TGF-β stimulation, and periodic short ligand exposures are sufficient to generate long-term signaling responses. Short-term TGF-β stimulation causes only transient pathway activation and can be terminated by ligand depletion. TGF-β-induced Smad2 phosphorylation is graded in the short-term but ultrasensitive (switch-like) in the long-term ( Figure 7). Additionally, cell growth arrest in response to TGF-β shows switch-like rather than graded behavior. Our modeling and experimental analyses suggest that ligand depletion is likely to be involved in sharpening a graded response into a switch-like response.

          Abstract

          Mammalian cells can decode the concentration of extracellular transforming growth factor-β (TGF-β) and transduce this cue into appropriate cell fate decisions. How variable TGF-β ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remain poorly understood. Using a combined experimental and mathematical modeling approach, we discovered that cells respond differently to continuous and pulsating TGF-β stimulation. The TGF-β pathway elicits a transient signaling response to a single pulse of TGF-β stimulation, whereas it is capable of integrating repeated pulses of ligand stimulation at short time interval, resulting in sustained phospho-Smad2 and transcriptional responses. Additionally, the TGF-β pathway displays different sensitivities to ligand doses at different time scales. While ligand-induced short-term Smad2 phosphorylation is graded, long-term Smad2 phosphorylation is switch-like to a small change in TGF-β levels. Correspondingly, the short-term Smad7 gene expression is graded, while long-term PAI-1 gene expression is switch-like, as is the long-term growth inhibitory response. Our results suggest that long-term switch-like signaling responses in the TGF-β pathway might be critical for cell fate determination.

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          Most cited references33

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          TGF-beta signal transduction.

          The transforming growth factor beta (TGF-beta) family of growth factors control the development and homeostasis of most tissues in metazoan organisms. Work over the past few years has led to the elucidation of a TGF-beta signal transduction network. This network involves receptor serine/threonine kinases at the cell surface and their substrates, the SMAD proteins, which move into the nucleus, where they activate target gene transcription in association with DNA-binding partners. Distinct repertoires of receptors, SMAD proteins, and DNA-binding partners seemingly underlie, in a cell-specific manner, the multifunctional nature of TGF-beta and related factors. Mutations in these pathways are the cause of various forms of human cancer and developmental disorders.
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            Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation.

            C Marshall (1995)
            A number of different intracellular signaling pathways have been shown to be activated by receptor tyrosine kinases. These activation events include the phosphoinositide 3-kinase, 70 kDa S6 kinase, mitogen-activated protein kinase (MAPK), phospholipase C-gamma, and the Jak/STAT pathways. The precise role of each of these pathways in cell signaling remains to be resolved, but studies on the differentiation of mammalian PC12 cells in tissue culture and the genetics of cell fate determination in Drosophila and Caenorhabditis suggest that the extracellular signal-regulated kinase (ERK-regulated) MAPK pathway may be sufficient for these cellular responses. Experiments with PC12 cells also suggest that the duration of ERK activation is critical for cell signaling decisions.
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              Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling.

              TGF-beta signals from the membrane to the nucleus through serine/threonine kinase receptors and their downstream effectors, termed SMAD proteins. The activated TGF-beta receptor induces phosphorylation of two such proteins, Smad2 and Smad3, which form hetero-oligomeric complex(es) with Smad4/DPC4 that translocate to the nucleus, where they then regulate transcriptional responses. However, the mechanisms by which the intracellular signals of TGF-beta are switched off are unclear. Here we report the identification of Smad7, which is related to Smad6. Transfection of Smad7 blocks responses mediated by TGF-beta in mammalian cells, and injection of Smad7 RNA into Xenopus embryos blocks activin/TGF-beta signalling. Smad7 associates stably with the TGF-beta receptor complex, but is not phosphorylated upon TGF-beta stimulation. TGFbeta-mediated phosphorylation of Smad2 and Smad3 is inhibited by Smad7, indicating that the antagonistic effect of Smad7 is exerted at this important regulatory step. TGF-beta rapidly induces expression of Smad7 mRNA, suggesting that Smad7 may participate in a negative feedback loop to control TGF-beta responses.
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                Author and article information

                Journal
                Mol Syst Biol
                Molecular Systems Biology
                Nature Publishing Group
                1744-4292
                2011
                24 May 2011
                24 May 2011
                : 7
                : 492
                Affiliations
                [1 ]simpleBIOSS Centre for Biological Signalling Studies and Center for Biological Systems Analysis (ZBSA), University of Freiburg , Freiburg, Germany
                [2 ]simpleDepartment of Chemistry and Biochemistry, University of Colorado , Boulder, CO, USA
                [3 ]simpleLudwig Institute for Cancer Research, BMC, Uppsala University , Uppsala, Sweden
                [4 ]simpleTheoretical Biophysics, Institute for Biology, Humboldt-Universität zu Berlin , Berlin, Germany
                [5 ]simpleDepartment of Medical Biochemistry and Microbiology, Science for Life Laboratory, BMC, Uppsala University , Uppsala, Sweden
                Author notes
                [a ]BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg 79104, Germany. Tel.: +49 761 203 97229; Fax: +49 761 203 97223; zhike.zi@ 123456bioss.uni-freiburg.de
                [b ]Department of Chemistry and Biochemistry, University of Colorado-Boulder, 215 UCB, Boulder, CO 80309-0215, USA. Tel.: +1 303 735 6161; Fax: +1 303 735 6161; Xuedong.Liu@ 123456colorado.edu
                [*]

                These authors contributed equally to this work

                Article
                msb201122
                10.1038/msb.2011.22
                3130555
                21613981
                fffdf07a-5f99-457f-9885-b6985290ebb0
                Copyright © 2011, EMBO and Macmillan Publishers Limited

                This is an open-access article distributed under the terms of the Creative Commons Attribution Noncommercial Share Alike 3.0 Unported License, which allows readers to alter, transform, or build upon the article and then distribute the resulting work under the same or similar license to this one. The work must be attributed back to the original author and commercial use is not permitted without specific permission.

                History
                : 27 September 2010
                : 07 April 2011
                Categories
                Article

                Quantitative & Systems biology
                mathematical model,ultrasensitivity,smad,tgf-β
                Quantitative & Systems biology
                mathematical model, ultrasensitivity, smad, tgf-β

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