Invasion control by a positive feedback loop mechanism Smad4-null cells
ABSTRACT: New findings demonstrate that transcriptional factors alternative to Smad4 can bind to Smad2/3 and mediate different transcriptional effects. In this study, we detected constitutively phosphorylation of Smad2/3 in Smad4-null pancreatic cancer cell line BxPC-3. Both pan-specific TGF-β-neutralizing antibody and specific TGF-β type I receptor (TβR-I) inhibitor, SB-431542, can decrease steady-state p-Smad2/3 levels. Moreover, exogenous TGF-β strongly stimulated translocation of phosphorylated Smad2/3 (p-Smad2/3) into the nucleus. Therefore, we identified TGF-β-responsive genes using genome-wide oligonucleotide microarrays and confirmed their dependency on Smad2/3 by the combination of RNA interference (RNAi) and microarray assay. The major finding from our microarray analysis was that of the 262 target genes seen to be regulated via TGF-β induction, 87 were differentially transcriptionally controlled by Smad2/3 signaling and 175 were Smad2/3-independent. Our results showed that integrin β6 was transcriptionally up-regulated via TGF-β induction in a Smad3-dependent manner, which was validated by real-time RT-PCR and western blot. We also provide evidence that αVβ6 integrin can activate TGF-β-Smad2/3 signaling. Thus, we for the first time suggest the positive feedback loop compose of TGF-β-Smad3 signaling and integrin β6. Functional analysis revealed that exogenous TGF-β can amplify the invasive property of Smad4-deficient pancreatic cancer cells; however, TGF-β-neutralizing antibody, specific TβR-I inhibitor, and anti-αVβ6 integrin antibody can reduce it. Therefore, integrin β6 mediated the invasion of BxPC-3 cells induced by TGF-β signaling. Keywords: cell type comparison Overall design: Large-scale microarray analysis was employed for comparison between the responses of wild-type BxPC-3 cell line and each knockdown cell line, with and without TGF-β induction, to identify target genes.
Project description:TGF-β signaling is known to be very much dependent on the formation of Smad2/3-Smad4 transcription regulatory complexes. However, the signaling functions of Smad2/3-Smad4 in TGF-β-induced responses are obscure as TGF-β also initiates a number of other signaling pathways. In this study, we systematically assessed the contribution of TGF-β-Smad2/3-Smad4 signaling to target gene transcription. Individual Smads were selectively knocked down in Hep3B cells by stable RNA interference (RNAi). We identified TGF-β-responsive genes using genome-wide oligonucleotide microarrays and confirmed their dependency on Smad2, Smad3 or Smad4 by the combination of RNAi and microarray assay. The major finding from our microarray analysis was that of the 2039 target genes seen to be regulated via TGF-β induction, 190 were differentially transcriptionally controlled by Smad2-Smad4 and Smad3-Smad4 signaling and the latter control mechanism appeared to be functionally more important. We also found evidence of competition between Smad2 and Smad3 for their activation when controlling the transcription of target genes. Keywords: cell type comparison Overall design: Large-scale microarray analysis was employed for comparison between the responses of wild-type Hep3B cell line and each knockdown cell line, with and without TGF-β induction, to identify target genes and any false positive genes.
Project description:The transforming growth factor beta (TGFβ) related signaling is one of the most important signaling pathways regulating early developmental events. Smad2 and Smad3 are structurally similar and it is mostly considered that they are equally important in mediating TGFβ signals. Here, we show that Smad3 is an insensitive TGFβ transducer as compared with Smad2. Smad3 preferentially localizes within the nucleus and is thus sequestered from membrane signaling. The ability of Smad3 in oligomerization with Smad4 upon agonist stimulation is also impaired given its unique linker region. Smad2 mediated TGFβ signaling plays a crucial role in epiblast development and patterning of three germ layers. However, signaling unrelated nuclear localized Smad3 is dispensable for TGFβ signaling-mediated epiblast specification, but important for early neural development, an event blocked by TGFβ/Smad2 signaling. Both Smad2 and Smad3 bind to the conserved Smads binding element (SBE), but they show nonoverlapped target gene binding specificity. We conclude that Smad2 and Smad3 possess differential sensitivities in relaying TGFβ signaling and have distinct roles in regulating early developmental events. GFP, GFP-Smad2 and GFP-Smad3 constitutively expressed Smad3-/- mouse ESCs were differentiated to day6 neuroepithelia and collected for Chip-Seq with an anti-GFP antibody.
Project description:The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS-domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit TGF-beta induced apoptosis. DACH1 repressed TGF-beta induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-beta responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dot-like structures. NCoR enhanced DACH1 repression and the repression of TGF-beta-induced AP-1 or Smad-signaling by DACH1 required the DACH1 DS domain. The DS-domain of DACH was sufficient for NCoR-binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-beta signaling by interacting with NCoR and Smad4.
Project description:TGF-beta treatment leads to SMAD1/5 phosphorylation. However, the ability of SMAD1/5 to bind chromatin downstream of TGF-beta signalling is unknown. We performed ChIP-sequencing for pSMAD1/5 and SMAD3 to identify binding sites for pSMAD1/5 upon TGF-beta stimulation and identified preferential pSMAD1/5 binding at SMAD1/5:SMAD4 consensus sites. Overall design: MDA-MB-231 cells untreated or treated with TGF-beta for 1 h and then examined with a SMAD3 or pSMAD1/5 antibody. The SMAD3 acts as a control as it is well known that SMAD3 is required for transcriptional regulation of many TGF-beta target genes.
Project description:Increasing evidence supports the idea that cancer cell plasticity promotes metastasis and tumor recurrence, resulting in patient mortality. While it is clear that the tumor microenvironment (TME) contributes to cancer cell plasticity, the specific TME factors most actively controlling plasticity remain largely unknown. Here, we performed a screen to identify TME cytokines and growth factors that promote epithelial-mesenchymal plasticity, and acquisition of cancer stem-cell (CSC) properties. Of 28 TME cytokines and growth factors tested, we identified Oncostatin M (OSM) as the most potent inducer of mesenchymal/CSC properties. OSM-induced plasticity was Signal Transducer and Activator of Transcription 3 (STAT3)-dependent, and also required a novel intersection with Transforming Growth Factor-β (TGF-β)/SMAD signaling. OSM/STAT3 activation promoted SMAD3 nuclear accumulation, DNA-binding, and induced SMAD3-dependent transcriptional activity. Suppression of TGF-β receptor activity or ablation of SMAD3 or SMAD4, but not SMAD2, strongly suppressed OSM/STAT3-mediated plasticity. Moreover, removal of OSM or inhibition of STAT3 or SMAD3 resulted in a marked reversion to a non-invasive, epithelial phenotype. We propose that, targeted blockade of the STAT3/SMAD3 axis in tumor cells may represent a novel therapeutic approach to prevent the plasticity required for metastatic progression and tumor recurrence. Overall design: Total RNA was isolated from sorted CD24+ 48R-shp16-shp53-MYC-RAS which were cultured under normal conditions (CD24+ NT) or in the presence of human recombinant OSM 10ng/mL (CD24+ OSM) or human recombinant TGF-b 10ng/mL (CD24+ TGF-b) for three weeks. Cells were treated at every medium change.
Project description:Transforming growth factor (TGF)-beta induces apoptosis of many types of cancer cells and acts as a tumor suppressor. We found lower expression of TGF-beta type II receptor (TbRII) in most of SCLC cells and tissues than in normal lung epithelial cells and normal lung tissues, respectively. In vitro cell growth and in vivo tumor formation were suppressed by TGF-beta-mediated apoptosis when the wild-type TbRII was overexpressed in SCLC cells. We therefore determined Smad2 and Smad3 (Smad2/3) binding sites in a SCLC cell line H345 stably expressing exogenous TbRII (H345-TbRII) to identify target genes of TGF-beta. Smad2 and Smad3 binding sites in H345-TbRII cells were determined by ChIP-seq (one sample analysis, without replicates).
Project description:Smad2/3 are transcription factors that engage in TGF-beta-induced transcription. Here we analyzed the effect of identified Smad2/3 binding sites to transcription. We used expression microarrays to compare the Smad2/3 binding sites identified by ChIP-chip to TGF-beta-induced gene expressions. Keywords: time course We also examined the effect of either ETS1/TFAP2A/SMAD2/SMAD3 siRNAs on TGF-beta-induced gene expression change. Overall design: HaCaT cells were treated with TGF-beta3 for Smad2/3 ChIP-chip or RNA extraction and hybridization on Affymetrix microarrays.
Project description:This model is from the article:
Quantitative analysis of transient and sustained transforming growth factor-β signaling dynamics.
Zhike Zi, Zipei Feng, Douglas A Chapnick, Markus Dahl, Difan Deng, Edda Klipp, Aristidis Moustakas & Xuedong Liu Molecular Systems Biology
2011 May 24;7:492. 21613981
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.
Developer of the model: Zhike Zi
Reference: Zi Z. et al., Quantitative Analysis of Transient and Sustained Transforming Growth Factor-beta Signaling Dynamics, Molecular Systems Biology, 2011
1. The global parameter that set the type of stimulation
(a) for sustained TGF-beta stimulation: set stimulation_type = 1.
(b) for single pulse of TGF-beta stimulation: set stimulation_type = 2.
parameter "single_pulse_duration" is for the duration of stimulation, for example,
single_pulse_duration = 0.5, for 0.5 min (30 seconds) of TGF-beta stimulation.
*Note: make sure that the time course cover the time point when the event is triggered.
(c) for single pulse of TGF-beta stimulation in COPASI
change the trigger of event "single_pulse_TGF_beta_washout"
"and(eq(stimulation_type, 2), eq(time, single_pulse_duration))" (for SBML-SAT)
"and(eq(stimulation_type, 2), gt(time, single_pulse_duration))" (for COPASI)
2. Notes for TGF-beta dose in terms of molecules per cell
(a) The following equation applies for conversion of TGF-beta dose in molecules per cell
TGF_beta_dose_mol_per_cell = initial TGF_beta_ex*1e-9*Vmed*6e23
(b) for standard experimental setup 1e6 cells in 2 mL medium
0.001 nM initial TGF_beta_ex is approximately equal to the dose of 1200 TGF-beta molecules/cell
0.050 nM initial TGF_beta_ex is approximately equal to the dose of 60000 TGF-beta molecules/cell
(c) For 1e6 cells in 10 mL medium, please change the initial compartment size of Vmed and the corresponding assignment rule for Vmed.
initial Vmed = 1e-8 (1e6 cells in 10 mL medium)
Vmed = 0.010/(1e6*exp(log(1.45)*time/1440)) (1e6 cells in 10 mL medium)
3. Please note that this model contains events and the medium compartment size is varied.
4. For the model simulation in SBML-SAT, please remove initialAssignments and save it as SBML Level 2 Verion 1 file.
Project description:Transforming growth factor (TGF)-β plays crucial roles in embryonic development and adult tissue homeostasis by eliciting various cellular responses in target cells. TGF-β signaling is principally mediated through receptor-activated Smad proteins, which regulate expression of target genes in cooperation with other DNA-binding transcriptionfactors (Smad cofactors). In this study, we found that the basic helix-loop-helix transcription factor Olig1 is a Smad cofactor involved in TGF-b-induced cell motility. Knockdown of Olig1 attenuated TGF-β-induced cell motility in chamber migration and wound healing assays. In contrast, Olig1 knockdown had no effect on bone morphogenetic protein-induced cell motility, TGF-β-induced cytostasis or epithelial-mesenchymal transition. Furthermore, we observed that cooperation of Smad2/3 with Olig1 is regulated by a peptidyl-prolyl cis/trans isomerase, Pin1. TGF-b-induced cell motility, induction of Olig1-regulated genes, and physical interaction between Smad2/3 and Olig1 were all inhibited after knockdown of Pin1, indicating a novel mode of regulation of Smad signaling. We also found that Olig1 interacts with the L3 loop of Smad3. Using a synthetic peptide corresponding to the L3 loop of Smad3, we succeeded in selectively inhibiting TGF-b-induced cell motility. These findings may lead to a new strategy for selective regulation of TGF-b-induced cellular responses. NMuMG cells were transfected with siRNAs (siControl, siOlig1 or siPin1) and treated with or without TGF-b for 1h. We compared genes affected by knockdown of Olig1 and that of Pin1.
Project description:Specific regulation of target genes by transforming growth factor-β (TGF-β) in a given cellular context is determined in part by transcription factors and cofactors that interact with the Smad complex. In the present study, we determined Smad2 and Smad3 (Smad2/3) binding regions in the promoters of known genes in HepG2 hepatoblastoma cells, and compared them to those in HaCaT epidermal keratinocytes to elucidate the mechanisms of cell type- and context-dependent regulation of transcription induced by TGF-β. Our results show that 81% of the Smad2/3 binding regions in HepG2 cells were not shared with those found in HaCaT cells. Hepatocyte nuclear factor 4α (HNF4α) is expressed in HepG2 cells, but not in HaCaT cells, and the HNF4α binding motif was identified as an enriched motif in the HepG2-specific Smad2/3 binding regions. ChIP-sequencing analysis of HNF4A binding regions under TGF-β stimulation revealed that 32.5% of the Smad2/3 binding regions overlapped HNF4A bindings. MIXL1 was identified as a new combinatorial target of HNF4A and Smad2/3, and both the HNF4A protein and its binding motif were required for the induction of MIXL1 by TGF-β in HepG2 cells. These findings generalize the importance of binding of HNF4A on Smad2/3 binding genomic regions for HepG2-specific regulation of transcription by TGF-β, and suggest that certain transcription factors expressed in a cell-type-specific manner play important roles in the transcription regulated by the TGF-β-Smad signaling pathway. HepG2 cells were treated with TGF-beta for 1.5 h or left untreated. anti-HNF4A ChIP-seq was performed. One lane was used for each sample.