RNA-Seq: Smad4-iECKO vs Smad4f/f P7 isolated retinal endothelial cells (iECKO=inducible, endothelial cell specific knockout)
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE116230 | GEO | 2018/12/18
REPOSITORIES: GEO
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Project description:To identify potential biological targets of the TGFβ pathway involved in AVM formation, we performed ChIP-sequencing experiments on BMP9 stimulated Ms1 endothelial cells (ECs). This data provides a comprehensive list of all SMAD4 binding sites in endothelial cells.
2018-12-18 | GSE115921 | GEO
Project description:Vascular networks form, remodel and mature under the influence of both fluid shear stress (FSS) and soluble factors. Physiological FSS promotes and maintains vascular stability via synergy with Bone Morphogenic Protein 9 (BMP9) and BMP10. Conversely, mutation of the BMP receptors ALK1, Endoglin or the downstream effector SMAD4 leads to Hereditary Hemorrhagic Telangiectasia (HHT), characterized by fragile and leaky arterial-venous malformations (AVMs). But how endothelial cells (EC) integrate FSS and BMP signals in vascular development and homeostasis, and how mutations give rise to vascular malformations is not well understood. Here, we aimed to elucidate the mechanism of synergy between fluid shear stress and SMAD signaling in vascular stability and its failure in HHT. We have now found that loss of Smad4 increases ECs’ sensitivity to flow by lowering the FSS set point with resulting AVMs exhibiting features of excessive flow-mediated morphological responses. Mechanistically, loss of Smad4 disinhibits flow-mediated Klf4-Tie2-PI3K/Akt signaling leading to increased EC proliferation and loss of arterial identity due to Klf4-mediated repression of cyclin dependent Kinase (CDK) inhibitors, CDKN2A and CDKN2B. Thus, AVMs caused by Smad4 deletion are characterized by chronic high flow remodeling with excessive EC proliferation and loss of arterial identity as triggering events.
2023-07-03 | GSE232443 | GEO
Project description:Purpose: Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant vascular disorder characterized by arteriovenous malformations (AVMs). Over 90% of HHT patients carry heterozygous loss-of-function mutations in Transforming Growth Factor-beta (TGFb) signaling components activin receptor-like kinase 1 (ACVRL1/ALK1), endoglin (ENG), or mothers against decapentaplegic homolog 4 (SMAD4). Brain AVMs can be lethal for HHT patients. Here, we use an inducible endothelial (EC)-specific mouse line to investigate brain AVMs characteristics in Alk1-, Eng-, and Smad4-HHT mouse models. Angiopientin-2 (Ang2) inhibition diminishes HHT associated cerebral vascular defects. We aim to identify the shared transcriptional changes in brain ECs between these three HHT types, and provide insights for transcriptional changes upon Ang2 inhibition. Methods: ECs were isolated from brains of both WT and HHT mutant mice at postnatal day 7 (P7) for Smad4 and Eng models, P6 for Alk1 model. Total RNA was extracted from isolated brain ECs and quantified using Qubit RNA High Sensitivity Assay Kit. RNA integrity was determined using the Bioanalyzer RNA 6000 Nano assay kit. RNA library construction was performed with the TruSeq RNA Library Prep Kit v2 from Illumina. The resulting mRNA library was quantified using Qubit dsDNA High Sensitivity Assay Kit and verified using the Bioanalyzer DNA1000 assay kit. Verified samples were sequenced using the NextSeq 500/550 High Output Kit v2.5 (150 Cycles) on a Nextseq 550 system. Sequenced reads were aligned to the mouse (mm10) reference genome with RNA-seq alignment tool (version 2.0.1). The aligned reads were used to quantify mRNA expression and determine differentially expressed genes using the RNA-seq Differential Expression tool (version 1.0.1). Both alignment and differential expression analysis were performed using the tools in the BaseSpace Sequence Hub. Results: We identified 5509, 5381, and 2663 differentially expressed genes in Smad4, Alk1, and Eng HHT models respectively. Also, Ang2 inhibition mainly funcioned through angiogenesis and cell migration processes to normalize cerebrovascular defects in Smad4-HHT model. Conclusions: A common but unique pro-angiogenic program among all HHT models included 14 consistent upregulated and 5 consistent downregulated angiogic genes. Also, Ang2 inhibition mainly funcioned through angiogenesis and cell migration processes to normalize cerebrovascular defects in Smad4-HHT model.
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Project description:RNA-sequencing of TGFβ treated empty vector (EV) or Smad4-expressing AKPS lines followed by GSEA revealed a number of gene sets overlapping with those obtained from analyses in reactive cholangiocytes. Hallmark gene sets MYC, E2F, G2M checkpoint, and oxidative phosphorylation were enriched with loss of Smad4, while TGFβ signaling, EMT, and TNFα were enriched with intact Smad4 signaling, suggesting conserved growth suppressive functions of Smad4 in primary cholangiocytes and advanced cancer.
2023-12-07 | GSE237679 | GEO
Project description:TGFβ is known to be a potent inducer of EMT, a process involved in tumor invasion. TIF1γ has been reported to participate to TGFβ signaling. In order to understand the role of TIF1γ in TGFβ signaling and its requirement for EMT, we analyzed the TGFβ1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ-silenced cells after TGFβ1 treatment, whereas Smad4 inactivation completely blocked this process. In support of these observations, microarray data show that the functions of several TIF1γ target genes can be linked to EMT. As a negative regulator of Smad4, TIF1γ could be critical for the regulation of TGFβ signaling. This work highlights the molecular relationship between TIF1γ and Smad4 in TGFβ1 signaling and EMT. Total mRNA extractions were performed for 11 samples from transfected HMEC-TR. Replicates are rimo1, 6; rimo3, 9; rimo2, 7; rimo 4, 10 and rimo 5, 11. Rimo 8 is a single experiment. All RNA extractions were obtained from two independent cell cultures excepted for rimo8. Rimo 1, 6 are replicate for ctrl-; Rimo 3, 9 are replicate for ctrl+; Rimo 2, 7 are replicate for SiTIF-; Rimo 4, 10 are replicate for SiTIF+; Rimo8 is SiSmad4-; and Rimo5, 11 are replicate for SiSmad4+. ctrl means that HMEC-TR were transfected with an SiRNA scramble. "siSmad4" means that HMEC-TR were transfected with an SiRNA anti Smad4. "siTIF" means that HMEC-TR were transfected with an SiRNA anti TIF1γ. "-" means that cells were grown without TGFβ. "+" means that cells were treated with 5 ng/ml TGFβ1 for 24h.
2011-05-26 | E-GEOD-28448 | biostudies-arrayexpress
Project description:We previously identified somatic activating KRAS mutations in a majority of human arteriovenous malformations (AVMs), using whole exome sequencing, which were enhanced in AVM endothelial cell fractions. We have now performed whole genome sequencing on AVM endothelial and non-endothelial cell fractions, as well as paired blood samples, in order to identify further somatic mutations.
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Project description:Deregulation of the transforming growth factor-β (TGFβ) signaling pathway in epithelial ovarian cancer has been reported, but the precise mechanism underlying disrupted TGFβ signaling in the disease remains unclear. We performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) to investigate genome-wide screening of TGFβ-induced SMAD4 binding in epithelial ovarian cancer. Following TGFβ stimulation of the A2780 epithelial ovarian cancer cell line, we identified 2,362 SMAD4 binding loci and 318 differentially expressed SMAD4 target genes. Comprehensive examination of SMAD4-bound loci, revealed four distinct binding patterns: 1) Basal; 2) Shift; 3) Stimulated Only; 4) Unstimulated Only. SMAD4-bound loci were primarily classified as either Stimulated only (74%) or Shift (25%), indicating that TGFβ-stimulation alters SMAD4 binding patterns in epithelial ovarian cancer cells compared to normal epithelial cells. Furthermore, based on gene regulatory network analysis, we determined that the TGFβ-induced SMAD4-dependent regulatory network was strikingly different in ovarian cancer compared to normal cells. Importantly, the TGFβ/SMAD4 target genes identified in the A2780 epithelial ovarian cancer cell line were predictive of patient survival, based on in silico mining of publically available patient data bases. In conclusion, our data highlight the utility of next generation sequencing technology to identify genome-wide SMAD4 target genes in epithelial ovarian cancer. The results link aberrant TGFβ/SMAD signaling to ovarian tumorigenesis. Furthermore, the identified SMAD4 binding loci, combined with gene expression profiling and in silico data mining of patient cohorts, may provide a powerful approach to determine potential gene signatures with biological and future translational research in ovarian and other cancers.
2011-07-01 | GSE27526 | GEO
Project description:Reduced representation bisulfite sequencing (RRBS) on the same TGFβ-treated AKPS lines (with EV or Smad4) revealed that Smad4 impacts DNA methylation (DNAme), with decreases in intergenic DNAme as the most impacted in our CCA models.
2023-12-07 | GSE237680 | GEO
Project description:TGFβ is known to be a potent inducer of EMT, a process involved in tumor invasion. TIF1γ has been reported to participate to TGFβ signaling. In order to understand the role of TIF1γ in TGFβ signaling and its requirement for EMT, we analyzed the TGFβ1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ-silenced cells after TGFβ1 treatment, whereas Smad4 inactivation completely blocked this process. In support of these observations, microarray data show that the functions of several TIF1γ target genes can be linked to EMT. As a negative regulator of Smad4, TIF1γ could be critical for the regulation of TGFβ signaling. This work highlights the molecular relationship between TIF1γ and Smad4 in TGFβ1 signaling and EMT.
2011-05-26 | GSE28448 | GEO
Project description:Transforming growth factor β (TGFβ) is a morphogenic protein that augments antiviral immunity by altering the functional properties of pathogen-specific memory CD8 T cells. During infection, TGFβ inhibits formation of effector (TEFF) and circulating memory CD8 T cells, while encouraging tissue-resident memory CD8 T cells (TRM) to settle in peripheral tissues. SMAD proteins are signaling intermediates that are used by members of the TGF cytokine family to modify gene expression. Using RNA-sequencing we determined that SMAD4 altered the transcriptional profile of antiviral CTLs during respiratory infection. Our data show that SMAD4 and TGFβ use alternate signaling pathways to cooperatively regulate a collection of genes that determine whether pathogen-specific memory CD8 T cells localize in peripheral or lymphoid tissues. During infection, SMAD4 acts independently of TGF to inhibit TRM development, while inducing genes that support formation of circulating memory CD8 T cells. The genes that are modulated by SMAD4 include several homing receptors (CD103, KLRG1 and CD62L) and transcription factors (Hobit and EOMES) that support memory formation.
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