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Myofibroblast de-differentiation proceeds via distinct transcriptomic and phenotypic transitions [RNA-Seq]

ABSTRACT: Myofibroblasts are the major cellular source of collagen, and their accumulation – via differentiation from fibroblasts and resistance to apoptosis – is a hallmark of tissue fibrosis. Clearance of myofibroblasts by de-differentiation and restoration of apoptosis sensitivity has the potential to reverse fibrosis. Prostaglandin E2 (PGE2) and mitogens such as fibroblast growth factor-2 (FGF2) have each been shown to de-differentiate myofibroblasts, but the resultant cellular phenotypes have neither been comprehensively characterized nor compared. Here we show that PGE2 elicited de-differentiation of human lung myofibroblasts via cAMP/PKA while FGF2 utilized MEK/ERK. The two mediators yielded transitional cells with distinct transcriptomes, with FGF2 promoting but PGE2 inhibiting proliferation and survival. The gene expression pattern in fibroblasts isolated from the lungs of mice undergoing resolution of experimental fibrosis resembled that of myofibroblasts treated with PGE2 in vitro. We conclude that myofibroblast de-differentiation can proceed via distinct programs exemplified by treatment with PGE2 and FGF2, with that occurring in vivo most closely resembling the former.

ORGANISM(S): Homo sapiens

PROVIDER: GSE163831 | GEO | 2021/03/23

REPOSITORIES: GEO

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Project description:Over the past decade, our laboratory developed a cat model of corneal wound healing after photorefractive keratectomy (PRK). Recently, we used this model in a combination of in vivo and in vitro approaches to show that myofibroblasts directly inhibit nerve regeneration in the wounded stroma, sub-basal plexus and epithelium. In vitro, two distinct phases to this inhibition have been observed: (1) initial slowing of neurite elongation via a releasable factor, and (2) neurite contact inhibition by myofibroblasts. For phase 1, transforming growth factor beta 1 (TGF-1) was shown to be the soluble, anti-neuritogenic factor released by myofibroblasts, and the specific signaling cascade it triggered to slow neurite elongation was identified. Ultimately, preventing myofibroblast differentiation suppressed both phase 1 and phase 2 inhibition, and resulted in faster nerve regeneration. However, in most cases of accidental injury, patients present after myofibroblast differentiation/fibrosis have occurred. The issue then is no longer preventing fibrosis, but rather overcoming its effects. We recently discovered that the synthetic PPAR ligand and Thiazolidinedione (TZD) drug, Troglitazone, stimulates myofibroblast de-differentiation in vitro and in vivo through a PPAR independent pathway. We now show that this TDZ’s ability to promote myofibroblast de-differentiation can be mimicked by UK-5099, a compound that spares PPAR, instead inhibiting a recently identified alternative target of TZDs: the mitochondrial pyruvate carrier (MPC). The recent literature and our preliminary results thus form a strong premise for refocusing our efforts on mitochondria, and critically testing the hypothesis that inhibiting the MPC facilitates myofibroblast de-differentiation, which together with metabolic remodeling in regenerating nerves can treat both fibrosis and overcome the blocked reinnervation inherent in its context. Work is ongoing to understand the metabolic changes that underlie the ability of TDZs to facilitate wound healing though mito-centric regulation of both myofibroblasts and corneal nerves. Here, we present the results of an RNA sequencing experiment designed to examine changes to the global transcriptome that occur as a result of TGF-β1-induced activation of corneal fibroblasts, and of TDZ- or UK5099-stimulated myofibroblast de-differentiation.

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: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.

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Myofibroblast de-differentiation proceeds via distinct transcriptomic and phenotypic transitions [RNA-Seq]

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