Smad2 and Smad3 binding sites in H345-TbRII cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Daizo Koinuma
PROVIDER: E-GEOD-63871 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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.
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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.
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Project description:TGF-βs regulate macrophage responses, by activating Smad2/3. We have previously demonstrated that macrophage-specific Smad3 stimulates phagocytosis and mediates anti-inflammatory macrophage transition in the infarcted heart. However, the role of macrophage Smad2 signaling in myocardial infarction remains unknown. We studied the role of macrophage-specific Smad2 signaling in the healing infarct, and we explored the basis for the distinct effects of Smad2 and Smad3. Infarct macrophages exhibited both Smad2 and Smad3 activation. In contrast to the effects of Smad3 loss, myeloid cell-specific Smad2 disruption had no effects on mortality, ventricular dysfunction and adverse remodeling, after myocardial infarction. Phagocytic removal of dead cells, macrophage and myofibroblast infiltration, collagen deposition, angiogenesis and scar remodeling were not affected by macrophage Smad2 loss. In isolated macrophages, TGF-β1, -β2 and -β3, activated both Smad2 and Smad3, whereas BMP6 triggered only Smad3 activation. Smad2 and Smad3 had similar patterns of nuclear translocation in response to TGF-β1. Smad3, and not Smad2, was the main mediator of transcriptional effects of TGF-β on macrophages and Smad3 loss resulted in enrichment of genes associated with RAR/RXR signaling, cholesterol biosynthesis and lipid metabolism. In conclusion, the in vivo and in vitro effects of TGF-β on macrophage function involve Smad3, and not Smad2.
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Project description:Smad family proteins transduce signals downstream of transforming growth factor-beta (TGF-beta) and are one of the factors that regulate target genes related to diseases affecting the skin. We here identified C2orf54, officially known as MAB21L4, as one of the most up-regulated targets of TGF-beta and Smad3 in a differentiated human progenitor epidermal keratinocyte, using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). Smad2 and Smad3 bind to the regulatory regions of the C2orf54 gene locus. We found that TGF-beta induced expression of a barrier protein involucrin (encoded by IVL gene), and transcriptional activity of the IVL promoter induced by TGF-beta was inhibited by siRNAs for C2orf54. Further analysis revealed that C2orf54 siRNAs also down-regulated the expression of several target genes of TGF-beta. C2orf54 protein located mainly in the cytosol, physically bound to Smad2 and Smad3, but did not inhibit the binding of Smad2 and Smad3 to the target genomic regions. These findings suggested that TGF-beta-induced C2orf54 up-regulates gene expression induced by Smads, possibly through its physical interaction with Smad proteins.
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Project description:Smad family proteins transduce signals downstream of transforming growth factor-beta (TGF-beta) and are one of the factors that regulate target genes related to diseases affecting the skin. We here identified C2orf54, officially known as MAB21L4, as one of the most up-regulated targets of TGF-beta and Smad3 in a differentiated human progenitor epidermal keratinocyte, using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). Smad2 and Smad3 bind to the regulatory regions of the C2orf54 gene locus. We found that TGF-beta induced expression of a barrier protein involucrin (encoded by IVL gene), and transcriptional activity of the IVL promoter induced by TGF-beta was inhibited by siRNAs for C2orf54. Further analysis revealed that C2orf54 siRNAs also down-regulated the expression of several target genes of TGF-beta. C2orf54 protein located mainly in the cytosol, physically bound to Smad2 and Smad3, but did not inhibit the binding of Smad2 and Smad3 to the target genomic regions. These findings suggested that TGF-beta-induced C2orf54 up-regulates gene expression induced by Smads, possibly through its physical interaction with Smad proteins.
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Project description: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
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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.
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