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Molecular analysis of the TGF-beta controlled gene expression program in chicken embryo dermal myofibroblasts

ABSTRACT: Myofibroblast is a specific type of mesenchymal cell characterized by synthesis of extracellular matrix and contractile activity. While it serves a beneficial function during tissue wound healing under physiological conditions, it can cause devastating damage to organs afflicted with fibrosis. Myofibroblasts are also present in tumor stroma and contribute actively to tumor growth and spreading. Chicken embryo dermal myofibroblasts (CEDM) represent a novel ex vivo model suitable for the analysis of myofibroblastic phenotype as they show strongly pronounced, uniform and self-sustained myofibroblastic phenotype that is stable in time. As myofibroblastic differentiation is controlled chiefly by TGF-beta signaling, the understanding of the differentiation program entails the determination of TGF-beta-regulated genes. To achieve such a goal, we performed oligonucleotide microarray analysis of CEDM cells treated with a selective TGFBR1 kinase inhibitor. Genes reported previously to be under the control of TGF-beta signaling in mammalian cells appeared among the affected genes also in CEDM cells and many so far unknown TGF-beta targets were revealed.

ORGANISM(S): Gallus gallus

PROVIDER: GSE28634 | GEO | 2011/04/15

SECONDARY ACCESSION(S): PRJNA138885

REPOSITORIES: GEO

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Project description:Background: Asthma is the most common chronic lung disease in children and young adults worldwide. Airway remodelling (including increased fibroblasts and myofibroblasts in airway walls due to chronic inflammation) differentiates asthmatic from non-asthmatic airways. The increase in airway fibroblasts and myofibroblasts occurs via epithelial to mesenchymal transition (EMT) where epithelial cells lose their tight junctions and are transdifferentiated to mesenchymal cells, with further increases in myofibroblasts occurring via fibroblast-myofibroblast transition (FMT). Transforming growth factor (TGF)-β is the central EMT- and FMT-inducing cytokine. In this study, we have used next generation sequencing to delineate the changes in the fibroblast transcriptome induced by TGF-β treatment in both the short term and after differentiation into myofibroblasts, to gain an understanding of the contribution of TGF-b induced transdifferentiation to the asthmatic phenotype. The data obtained from RNAseq analysis was confirmed by quantitative PCR (qPCR). Results: As expected, we found that genes coding for intermediates in the TGF-β signalling pathways (SMADs) were differentially expressed after treatment, and genes involved in cytoskeletal pathways (FN1, LAMA, ITGB1) were differentially expressed in myofibroblasts compared to fibroblasts. Importantly, genes that were previously shown to be changed in asthmatic lungs (ADAMTS1, DSP, TIMPs, MMPs) were differentially expressed in myofibroblasts, strongly suggesting that TGF-β mediated differentiation of fibroblasts to myofibroblasts may underlie important changes in the asthmatic airway. We also identified new signalling pathways (AKT, PTEN) that are changed in myofibroblasts compared to fibroblasts. Conclusion: We have found a significant number of genes that are altered after differentiation of fibroblasts into myofibroblasts by TGF-β treatment, many of which were expected or predicted. However, we also identified novel genes and pathways that were affected after treatment of fibroblasts with TGF-β, which suggests additional pathways that that are activated during the transition between fibroblasts and myofibroblasts, and may contribute to the asthma phenotype.

Project description:Tumor microenvironment contains various components including cancer cells, tumor vessels, and cancer associated fibroblasts (CAFs), comprising of tumor-promoting myofibroblasts and tumor-suppressing fibroblasts. Multiple lines of evidence indicated that transforming growth factor-β (TGF-β) induces the formation of myofibroblasts and other types of mesenchymal (non-myofibroblastic) cells from endothelial cells. Recent reports showed that fibroblast growth factor 2 (FGF2) modulates TGF-β-induced mesenchymal transition of endothelial cells, but the molecular mechanisms regarding the signals that control the transcriptional networks during the formation of different groups of fibroblasts remain largely unclear. Here, we studied the roles of FGF2 during the regulation of TGF-β-induced mesenchymal transition of tumor endothelial cells (TECs). We demonstrated that auto/paracrine FGF signals in TECs inhibit TGF-β-induced endothelial-to-myofibroblast transition (End-MyoT), leading to suppressed formation of contractile myofibroblast cells, but on the other hand can also collaborate with TGF-β in promoting the formation of active fibroblastic cells which have migratory and proliferative properties. FGF2 modulated TGF-β-induced formation of myofibroblastic and non-myofibroblastic cells from TECs via transcriptional regulation of the array of various mesenchymal markers and growth factors. Furthermore, we observed that TECs treated with TGF-β were more competent in promoting in vivo tumor growth than TECs treated with TGF-β and FGF2. Mechanistically, we showed that Elk1 mediated this FGF2-induced inhibition of End-MyoT via inhibition of TGF-β-induced transcriptional activation of α-SMA promoter by myocardin-related transcription factor (MRTF)-A. Our data suggest that TGF-β and FGF2 oppose and cooperate with each other during the formation of myofibroblastic and non-myofibroblastic cells from TECs to determine the characteristics of the mesenchymal cells in tumor microenvironment. Identification of marker genes for TGF-β-induced endothelial-to-myofibroblast transition

Project description:As a group, fibroproliferative disorders of the lung, liver, kidney, heart, vasculature and integument are common, progressive and refractory to therapy. They can emerge following toxic insults, but are frequently idiopathic. Their enigmatic propensity to resist therapy and progress to organ failure has focused attention on the myofibroblast – the primary effector of the fibroproliferative response. A central unanswered question is whether these myofibroblasts have acquired a distinct pathological phenotype - or whether they are normal myofibroblasts with a pathological phenotype that depends upon residing in a sea of pro-fibrotic cytokines and an abnormal extracellular matrix. Using a systems approach to study this question in a prototype fibrotic disease, Idiopathic Pulmonary Fibrosis (IPF); here we show organized changes in the gene expression pathway of primary lung myofibroblasts that persist for up to 9 sub-cultivations in vitro. When comparing IPF and control myofibroblasts in a 3-dimensional type I collagen matrix, more genes differed at the level of ribosome recruitment than at the level of transcript abundance, indicating pathological translational control as a major characteristic of IPF myofibroblasts. To determine the effect of matrix state on translational control, myofibroblasts were permitted to contract the matrix. Ribosome recruitment in control myofibroblasts was relatively stable. In contrast, IPF cells manifested large alterations in the ribosome recruitment pattern. Pathological studies suggest an epithelial origin for IPF myofibroblasts through the epithelial to mesenchymal transition (EMT). In accord with this, we found systems-level indications for TGF?b -driven EMT as one source of IPF myofibroblasts. Keywords: cell type comparison Comparison of lung myofibroblasts from patients with Idiopatic Pulmonary Fibrosis (IPF, 6 donors) to control (6 donors) in contractile and non-contractile matrices using both total and polyribosomal RNA

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Molecular analysis of the TGF-beta controlled gene expression program in chicken embryo dermal myofibroblasts

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