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Isoform-specific effects of transforming growth factor ? on endothelial-to-mesenchymal transition.

ABSTRACT: Endothelial-to-mesenchymal transition (EndMT) was first reported in the embryogenesis. Recent studies show that EndMT also occurs in the disease progression of atherosclerosis, cardiac and pulmonary fibrosis, pulmonary hypertension, diabetic nephropathy, and cancer. Although transforming growth factor ? (TGF?) is crucial for EndMT, it is not clear which isoform elicits a predominant effect. The current study aims to directly compare the dose-dependent effects of TGF?1, TGF?2, and TGF?3 on EndMT and characterize the underlying mechanisms. In our results, all three TGF? isoforms induced EndMT in human microvascular endothelial cells after 72?hr, as evidenced by the increased expression of mesenchymal markers N-cadherin and ?-smooth muscle actin as well as the decreased expression of endothelial nitric oxide synthase. Interestingly, the effect of TGF?2 was the most pronounced. At 1?ng/ml, only TGF?2 treatment resulted in significantly increased phosphorylation (activation) of Smad2/3 and p38-MAPK and increased expression of mesenchymal transcription factors Snail and FoxC2. Intriguingly, we observed that treatment with 1?ng/ml TGF?1 and TGF?3, but not TGF?2, resulted in an increased expression of TGF?2, thus indicating that EndMT with TGF?1 and TGF?3 treatments was due to the secondary effects through TGF?2 secretion. Furthermore, silencing TGF?2 using small interfering RNA blunted the expression of EndMT markers in TGF?1- and TGF?3-treated cells. Together, our results indicate that TGF?2 is the most potent inducer of EndMT and that TGF?1- and TGF?3-induced EndMT necessitates a paracrine loop involving TGF?2.

SUBMITTER: Sabbineni H

PROVIDER: S-EPMC6415927 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Isoform-specific effects of transforming growth factor β on endothelial-to-mesenchymal transition.

Sabbineni Harika H Verma Arti A Somanath Payaningal R PR

Journal of cellular physiology 20180601 11

Endothelial-to-mesenchymal transition (EndMT) was first reported in the embryogenesis. Recent studies show that EndMT also occurs in the disease progression of atherosclerosis, cardiac and pulmonary fibrosis, pulmonary hypertension, diabetic nephropathy, and cancer. Although transforming growth factor β (TGFβ) is crucial for EndMT, it is not clear which isoform elicits a predominant effect. The current study aims to directly compare the dose-dependent effects of TGFβ1, TGFβ2, and TGFβ3 on EndMT ...[more]

PMID: 29856065

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Project description:BackgroundDuring metastasis, cancer cells undergo epithelial-mesenchymal transition (EMT) in response to transforming growth factor-β (TGF-β), which is abundant in the tumor microenvironment, and acquire invasive and metastatic potentials. Metastasis to distant organs requires intravascular invasion and extravasation of cancer cells, which is accompanied by the disruption of the adhesion between vascular endothelial cells. Cancer cell-derived extracellular vesicles (EVs) have been suggested to induce the destabilization of normal blood vessels at the metastatic sites. However, the roles of EVs secreted from cancer cells that have undergone EMT in the destabilization of blood vessels remain to be elucidated. In the present study, we characterized EVs secreted by oral cancer cells undergoing TGF-β-induced EMT and elucidated their effects on the characteristics of vascular endothelial cells.MethodsInduction of EMT by TGF-β in human oral cancer cells was assessed using quantitative RT-PCR (qRT-PCR) and immunocytochemistry. Oral cancer cell-derived EVs were isolated from the conditioned media of oral cancer cells that were treated with or without TGF-β using ultracentrifugation, and characterized using nanoparticle tracking analysis and immunoblotting. The effects of EVs on human umbilical artery endothelial cells were examined by qRT-PCR, cellular staining, and permeability assay. The significant differences between means were determined using a t-test or one-way analysis of variance with Tukey's multiple comparisons test.ResultsOral cancer cells underwent EMT in response to TGF-β as revealed by changes in the expression of epithelial and mesenchymal cell markers at both the RNA and protein levels. Oral cancer cells treated with TGF-β showed increased EV production and altered EV composition when compared with untreated cells. The EVs that originated from cells that underwent EMT by TGF-β induced endothelial-mesenchymal transition, which was characterized by the decreased and increased expression of endothelial and mesenchymal cell markers, respectively. EVs derived from oral cancer cells also induced intercellular gap formation which led to the loss of endothelial cell barrier stability.ConclusionsEVs released from oral cancer cells that underwent TGF-β-induced EMT target endothelial cells to induce vascular destabilization. Detailed characterization of oral cancer-derived EVs and factors responsible for EV-mediated vascular instability will lead to the development of agents targeting metastasis.

Project description:The tumor microenvironment contains various components, including cancer cells, tumor vessels, and cancer-associated fibroblasts, the latter of which are comprised of tumor-promoting myofibroblasts and tumor-suppressing fibroblasts. Multiple lines of evidence indicate 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 show that fibroblast growth factor 2 (FGF2) modulates TGF-β-induced mesenchymal transition of endothelial cells, but the molecular mechanisms behind the signals that control 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 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 FGF2-induced inhibition of End-MyoT via inhibition of TGF-β-induced transcriptional activation of α-smooth muscle actin promoter by myocardin-related transcription factor-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, which in turn determines the characteristics of mesenchymal cells in the tumor microenvironment.

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Isoform-specific effects of transforming growth factor ? on endothelial-to-mesenchymal transition.

Publications

Isoform-specific effects of transforming growth factor β on endothelial-to-mesenchymal transition.

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