TGF-beta/Smad3-dependent gene expression analysis in naive CD4 T cells
ABSTRACT: Investigation of transcript level modulation in unstimulated and TGF-beta treated (with or without superimposed T-cell receptor and CD28 stimulation) naive CD4 T cells from wild type or Smad3-deficient littermate mice. Smad3 is a critical signaling molecule and transcription factor downstream of TGF-beta and mediates several of the TGF-beta dependent tolerogenic effects in T cells. This study was undertaken to unveil the transcriptionnal program controled by the TGF-b/Smad3 axis. Microarray study using RNA recovered after 6 hours of culture in either serum free media, serum-free media + TGF-beta (2.5ng/ml) or serum-free media + TGF-beta and anti-CD3e and anti-CD28 stimulation (3 conditions). Naive CD4 T cells (TCRb+, CD4+, CD62L+ and CD44-) were sorted from either wild type or Smad3 deficient littermates and submitted to the 3 culture conditions. Three biological replicates were obtained (each from at least 2 different mice). Thus a total of 18 Nimblegen 365K chip were used.
Project description:Imbalances in glucose and energy homeostasis are at the core of the worldwide epidemic of obesity and diabetes. Here, we illustrate an important role of the TGF-beta/Smad3 signaling pathway in regulating glucose and energy homeostasis. Smad3 deficient mice are protected from diet-induced obesity and diabetes. Interestingly, the metabolic protection is accompanied by Smad3-/- white adipose tissue acquiring the bioenergetic and gene expression profile of brown fat/skeletal muscle. Smad3-/- adipocytes demonstrate a marked increase in mitochondrial biogenesis, with a corresponding increase in basal respiration, and Smad3 acts as a repressor of PGC-alpha1 expression. We observe significant correlation between TGF-beta1 levels and adiposity in rodents and humans. Further, systemic blockade of TGF-beta1 signaling protects mice from obesity, diabetes and hepatic steatosis. Together, these results demonstrate that TGF-beta signaling regulates glucose tolerance and energy homeostasis and suggest that modulation of TGF-beta1 activity might be an effective treatment strategy for obesity and diabetes. Smad3-/- and WT mice were fed with regular diet (RD) and high fat diet (HFD), and diet induced obese (DIO) mice were treated with IgG and anti-TGF-b1 antibody
Project description:To find potential microRNA links between Smad3 and E-cadherin, we characterized the microRNA profiles of two gastric cancer cell lines: SNU484-LPCX, which does not express Smad3, and SNU484-Smad3, in which Smad3 is overexpressed. We found that miR-200 families, among other differentially expressed miRNAs, are overexpressed in SNU484-Smad3. Through subsequent studies, including silencing of Smad3 in SNU484-Smad3 and expression profiling of epithelial-mesenchymal markers and ZEB1/2, known repressors of E-cadherin, we found that Smad3 regulates miR-200 families at the transcriptional level, which regulate ZEB 1/2, known transcriptional repressors of E-cadherin, at the post-transcriptional level. This represents an important link between the TGF-beta signaling pathway and post-transcriptional regulation by miRNAs. Examination of small RNA expression in 2 different cell lines (SNU484-LPCX, SNU484-Smad3).
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:TGF-betas have complex roles in tumorigenesis, with context-dependent effects that can either suppress or promote tumor progression. Our goal was to use integrated genomic approaches in a model of human breast cancer progression to identify core TGF-beta-regulated genes that specifically reflect the tumor suppressor activity of TGF-beta. The model consisted of the non-tumorigenic MCF10A (“M1”), the premalignant MCF10AT1k.cl2 (“M2”), the early malignant MCF10Ca1h (“M3”) and the highly malignant, metastatic MCF10Ca1a.cl1 (“M4”) cell lines. We have previously shown that tumor suppressor activity of TGF-beta is dependent on Smad3, and is lost in M4 cells. To identify how TGF-beta/Smad3 targets change with cancer progression, we performed promoter-wide Smad3 ChIP-chip on all four cell lines of the breast cancer progression model (M1-M4), following treatment with TGF-beta or vehicle control. Overall design: Subconfluent cultures of the four cell lines (M1-M4) were serum-starved for 16 h and then treated with 5ng/ml of TGF-beta1 (plus condition) or vehicle alone (minus condition) for 1hour. Smad3 ChIP was performed using the Abcam #28379 anti-Smad3 antibody. Four biological replicates were performed for each condition (plus and minus) for M1 and M2 cells, and two biological replicates for each condition for M3 and M4 cells; total of 24 samples.
Project description:Based on studies in knockout mice, several inhibitory factors such as TGF-beta, IL-10, or CTLA-4 have been implicated as gate keepers of adaptive immune responses. Lack of these inhibitory molecules leads to massive inflammatory responses mainly mediated by activated T cells. In humans, the integration of these inhibitory signals for keeping T cells at a resting state is less well understood. To elucidate this regulatory network we assessed early genome-wide transcriptional changes during serum deprivation in human mature CD4+ T cells. The most striking observation was a "TGF-beta loss signature" defined by downregulation of many known TGF-beta target genes. Moreover, numerous novel TGF-beta target genes were identified that are under the suppressive control of TGF-beta. Expression of these genes was upregulated once TGF-beta signaling was lost during serum deprivation and again suppressed upon TGF-beta reconstitution. Constitutive TGF-beta signaling was corroborated by demonstrating phosphorylated SMAD2/3 in resting human CD4+ T cells in situ, which were dephosphorylated during serum deprivation and re-phosphorylated by minute amounts of TGF-beta. Loss of TGF-beta signaling was particularly important for T cell proliferation induced by low-level T cell receptor and costimulatory signals. We suggest TGF-beta to be the most prominent factor actively keeping human CD4+ T cells at a resting state. Keywords: time course, dose response Overall design: Early genome-wide transcriptional changes during serum deprivation in human mature CD4+ T cells assessed using Affymetrix HGU133A and Illumina Sentrix BeadChip arrays.
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:Mitogen-activated protein kinases (MAPKs) are key mediators of the T cell receptor (TCR) signals but their roles in T helper (Th) cell differentiation are unclear. Here we showed that the MAPK kinase kinases MEKK2 (encoded by Map3k2) and MEKK3 (encoded by Map3k3),negatively regulated transforming growth factor-beta (TGF-beta)-mediated Th cell differentiation.Map3k2-/-Map3k3Lck-Cre/- mice showed an abnormal accumulation of regulatory T (Treg) and Th17 cells in the periphery, consistent with Map3k2-/-Map3k3Lck-Cre/- naïve CD4+ T cells’ differentiation into Treg and Th17 cells with a higher frequency than wild-type (WT) cells after TGF-beta stimulation in vitro. In addition, Map3k2-/-Map3k3Lck-Cre/- mice developed more severe experimental autoimmune encephalomyelitis. Map3k2-/-Map3k3Lck-Cre/- T cells exhibited impaired phosphorylation of the SMAD2 and SMAD3 proteins at their linker regions, which negatively regulated the TGF-beta responses in T cells. Thus, the crosstalk between TCR-induced MAPK and the TGF-beta signaling pathways is important in regulating Th cell differentiation. CD4+CD62L-CD44+ cells were FACS sorted from C57BL/6 Lck-Cre MEKK2 KO MEKK3F/-(dKO) or C57BL/6 WT mice
Project description:The aim of this study is to identify SMAD3 binding targets affected by the TGFb1/SMAD3 signal transduction on a genome-wide scale Formaldehyde cross-linked, sonicated chromatin was prepared from non-stimulated A549 cell line and stimulated with TGFb1. Chromatin immunoprecipated with anti-SMAD3 antibody is labeled with Cy5 and mock IP (with anti-FLAG antibody) is labeled with Cy3 and co-hybridized on Agilent Human Promoter Set arrays. Two biological replicates were performed.
Project description:Mitogen-activated protein kinases (MAPKs) are key mediators of the T cell receptor (TCR) signals but their roles in T helper (Th) cell differentiation are unclear. Here we showed that the MAPK kinase kinases MEKK2 (encoded by Map3k2) and MEKK3 (encoded by Map3k3),negatively regulated transforming growth factor-beta (TGF-beta)-mediated Th cell differentiation.Map3k2-/-Map3k3Lck-Cre/- mice showed an abnormal accumulation of regulatory T (Treg) and Th17 cells in the periphery, consistent with Map3k2-/-Map3k3Lck-Cre/- naïve CD4+ T cells' differentiation into Treg and Th17 cells with a higher frequency than wild-type (WT) cells after TGF-beta stimulation in vitro. In addition, Map3k2-/-Map3k3Lck-Cre/- mice developed more severe experimental autoimmune encephalomyelitis. Map3k2-/-Map3k3Lck-Cre/- T cells exhibited impaired phosphorylation of the SMAD2 and SMAD3 proteins at their linker regions, which negatively regulated the TGF-beta responses in T cells. Thus, the crosstalk between TCR-induced MAPK and the TGF-beta signaling pathways is important in regulating Th cell differentiation. Overall design: CD4+CD62L-CD44+ cells were FACS sorted from C57BL/6 Lck-Cre MEKK2 KO MEKK3F/-(dKO) or C57BL/6 WT mice