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Pulmonary vascular endothelial function may be impaired by oxidative stress in endotoxemia-derived acute lung injury. Growing evidence suggests that endothelial-to-mesenchymal transition (EndMT) could play a pivotal role in various respiratory diseases; however, it remains unclear whether EndMT participates in the injury/repair process of septic acute lung injury. Here, we analyzed lipopolysaccharide (LPS)-treated mice whose total number of pulmonary vascular endothelial cells (PVECs) transiently decreased after production of reactive oxygen species (ROS), while the population of EndMT-PVECs significantly increased. NAD(P)H oxidase inhibition suppressed EndMT of PVECs. Most EndMT-PVECs derived from tissue-resident cells, not from bone marrow, as assessed by mice with chimeric bone marrow. Bromodeoxyuridine-incorporation assays revealed higher proliferation of capillary EndMT-PVECs. In addition, EndMT-PVECs strongly expressed c-kit and CD133. LPS loading to human lung microvascular endothelial cells (HMVEC-Ls) induced reversible EndMT, as evidenced by phenotypic recovery observed after removal of LPS. LPS-induced EndMT-HMVEC-Ls had increased vasculogenic ability, aldehyde dehydrogenase activity, and expression of drug resistance genes, which are also fundamental properties of progenitor cells. Taken together, our results demonstrate that LPS induces EndMT of tissue-resident PVECs during the early phase of acute lung injury, partly mediated by ROS, contributing to increased proliferation of PVECs.
Copyright © 2016 the American Physiological Society.
Dormant or slow-cycling disseminated tumor cells (DTCs) in bone marrow (BM) are resistant to conventional therapy in various cancers including head and neck squamous cell carcinoma (HNSCC), although the molecular mechanisms remain largely unknown. This study aimed to identify the intrinsic molecular mechanisms underlying drug resistance in BM-DTCs. We used in vivo selection of the human HNSCC cell line HEp3, which mimics non-proliferative BM-DTCs in mice, to establish BM-DTC-derived (BM-HEp3) and lung metastases-derived (Lu-HEp3) sublines. Both sublines had higher migration activity and shortened survival in a murine xenograft model compared with parental (P-HEp3) cells. Slow-cycling BM-HEp3 cells had intrinsically enhanced cisplatin resistance compared with Lu-HEp3 cells, which also manifested this resistance but proliferated rapidly. The drug resistance and slow-cycling state of BM-HEp3 cells depended on enhanced positive feedback of the signaling axis of stromal cell-derived factor-1 (SDF-1)-C-X-C chemokine receptor-4 (CXCR4) via their overexpression. Interestingly, BM-DTCs highly expressed transforming growth factor-beta 2 (TGF-β2) to maintain SDF-1-CXCR4 overexpression. Inhibition of SDF-1-CXCR4 signaling by down-regulating TGF-β2 fully reversed the drug resistance of BM-HEp3 cells via reactivation of cell proliferation. These data suggest that the intrinsic TGF-β2-triggered SDF-1-CXCR4 signaling axis is crucial for drug resistance dependent on a slow-cycling state in BM-DTCs.
Preterm infants may be at risk of necrotizing enterocolitis (NEC) due to deficiency of transforming growth factor-β 2 (TGF-β(2)) in the developing intestine. We hypothesized that low epithelial TGF-β(2) expression in preterm intestine and during NEC results from diminished autocrine induction of TGF-β(2) in these cells. Premature baboons delivered at 67% gestation were treated per current norms for human preterm infants. NEC was diagnosed by clinical and radiological findings. Inflammatory cytokines, TGF-β(2), Smad7, Ski, and strawberry notch N (SnoN)/Ski-like oncoprotein (SKIL) was measured using quantitative reverse transcriptase-polymerase chain reaction, immunoblots, and immunohistochemistry. Smad7 effects were examined in transfected IEC6 intestinal epithelial cells in vitro. Findings were validated in archived human tissue samples of NEC. NEC was recorded in seven premature baboons. Consistent with existing human data, premature baboon intestine expressed less TGF-β(2) than term intestine. TGF-β(2) expression was regulated in epithelial cells in an autocrine fashion, which was interrupted in the premature intestine and during NEC due to increased expression of Smad7. LPS increased Smad7 binding to the TGF-β(2) promoter and was associated with dimethylation of the lysine H3K9, a marker of transcriptional silencing, on the nucleosome of TGF-β(2). Increased Smad7 expression in preterm intestine was correlated with the deficiency of SnoN/SKIL, a repressor of the Smad7 promoter. Smad7 inhibits autocrine expression of TGF-β(2) in intestinal epithelial cells in the normal premature intestine and during NEC. Increased Smad7 expression in the developing intestine may be due to a developmental deficiency of the SnoN/SKIL oncoprotein.
In addition to parenchymal fibrosis, fibrotic remodeling of the distal airways has been reported in interstitial lung diseases. Mechanisms of airway wall remodeling, which occurs in a variety of chronic lung diseases, are not well defined and current animal models are limited. The authors quantified airway remodeling in lung sections from subjects with idiopathic pulmonary fibrosis (IPF) and controls. To investigate intratracheal bleomycin as a potential animal model for fibrotic airway remodeling, the authors evaluated lungs from C57BL/6 mice after bleomycin treatment by histologic scoring for fibrosis and peribronchial inflammation, morphometric evaluation of subepithelial connective tissue volume density, TUNEL (terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling) assay, and immunohistochemistry for transforming growth factor β1 (TGFβ1), TGFβ2, and the fibroblast marker S100A4. Lung mechanics were determined at 3 weeks post bleomycin. IPF lungs had small airway remodeling with increased bronchial wall thickness compared to controls. Similarly, bleomycin-treated mice developed dose-dependent airway wall inflammation and fibrosis and greater airflow resistance after high-dose bleomycin. Increased TUNEL(+) bronchial epithelial cells and peribronchial inflammation were noted by 1 week, and expression of TGFβ1 and TGFβ2 and accumulation of S100A4(+) fibroblasts correlated with airway remodeling in a bleomycin dose-dependent fashion. IPF is characterized by small airway remodeling in addition to parenchymal fibrosis, a pattern also seen with intratracheal bleomycin. Bronchial remodeling from intratracheal bleomycin follows a cascade of events including epithelial cell injury, airway inflammation, profibrotic cytokine expression, fibroblast accumulation, and peribronchial fibrosis. Thus, this model can be utilized to investigate mechanisms of airway remodeling.
Coronary vessel development requires transfer of mesothelial cells to the heart surface to form the epicardium where some cells subsequently undergo epithelial-mesenchymal transformation (EMT) and invade the subepicardial matrix. Tgfbr3(-/-) mice die due to failed coronary vessel formation associated with decreased epicardial cell invasion but the mediators downstream of TGFβR3 are not well described. TGFβR3-dependent endocardial EMT stimulated by either TGFβ2 or BMP-2 requires activation of the Par6/Smurf1/RhoA 1pathway where Activin Receptor Like Kinase (ALK5) signals Par6 to act downstream of TGFβ to recruit Smurf1 to target RhoA for degradation to regulate apical-basal polarity and tight junction dissolution. Here we asked if this pathway was operant in epicardial cells and if TGFβR3 was required to access this pathway. Targeting of ALK5 in Tgfbr3(+/+) cells inhibited loss of epithelial character and invasion. Overexpression of wild-type (wt) Par6, but not dominant negative (dn) Par6, induced EMT and invasion while targeting Par6 by siRNA inhibited EMT and invasion. Overexpression of Smurf1 and dnRhoA induced loss of epithelial character and invasion. Targeting of Smurf1 by siRNA or overexpression of constitutively active (ca) RhoA inhibited EMT and invasion. In Tgfbr3(-/-) epicardial cells which have a decreased ability to invade collagen gels in response to TGFβ2, overexpression of wtPar6, Smurf1, or dnRhoA had a diminished ability to induce invasion. Overexpression of TGFβR3 in Tgfbr3(-/-) cells, followed by siRNA targeting of Par6 or Smurf1, diminished the ability of TGFβR3 to rescue invasion demonstrating that the Par6/Smurf1/RhoA pathway is activated downstream of TGFβR3 in epicardial cells.
Copyright © 2011 Elsevier Inc. All rights reserved.
An early event in heart valve formation is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endothelial cells in specific regions of the heart tube, the endocardial cushions. The Type III TGFβ receptor (TGFβR3) is required for TGFβ2- or BMP-2-stimulated EMT in atrioventricular endocardial cushion (AVC) explants in vitro but the mediators downstream of TGFβR3 are not well described. Using AVC and ventricular explants as an in vitro assay, we found an absolute requirement for specific TGFβR3 cytoplasmic residues, GAIP-interacting protein, C terminus (GIPC), and specific Activin Receptor-Like Kinases (ALK)s for TGFβR3-mediated EMT when stimulated by TGFβ2 or BMP-2. The introduction of TGFβR3 into nontransforming ventricular endocardial cells, followed by the addition of either TGFβ2 or BMP-2, results in EMT. TGFβR3 lacking the entire cytoplasmic domain, or only the 3C-terminal amino acids that are required to bind GIPC, fails to support EMT in response to TGFβ2 or BMP-2. Overexpression of GIPC in AVC endocardial cells enhanced EMT while siRNA-mediated silencing of GIPC in ventricular cells overexpressing TGFβR3 significantly inhibited EMT. Targeting of specific ALKs by siRNA revealed that TGFβR3-mediated EMT requires ALK2 and ALK3, in addition to ALK5, but not ALK4 or ALK6. Taken together, these data identify GIPC, ALK2, ALK3, and ALK5 as signaling components required for TGFβR3-mediated endothelial cell EMT.
Copyright Â© 2011. Published by Elsevier Inc.
The epicardium is a major contributor of the cells that are required for the formation of coronary vessels. Mice lacking both copies of the gene encoding the Type III Transforming Growth Factor β Receptor (TGFβR3) fail to form the coronary vasculature, but the molecular mechanism by which TGFβR3 signals coronary vessel formation is unknown. We used intact embryos and epicardial cells from E11.5 mouse embryos to reveal the mechanisms by which TGFβR3 signals and regulates epicardial cell behavior. Analysis of E13.5 embryos reveals a lower rate of epicardial cell proliferation and decreased epicardially derived cell invasion in Tgfbr3(-/-) hearts. Tgfbr3(-/-) epicardial cells in vitro show decreased proliferation and decreased invasion in response to TGFβ1 and TGFβ2. Unexpectedly, loss of TGFβR3 also decreases responsiveness to two other important regulators of epicardial cell behavior, FGF2 and HMW-HA. Restoring full length TGFβR3 in Tgfbr3(-/-) cells rescued deficits in invasion in vitro in response TGFβ1 and TGFβ2 as well as FGF2 and HMW-HA. Expression of TGFβR3 missing the 3 C-terminal amino acids that are required to interact with the scaffolding protein GIPC1 did not rescue any of the deficits. Overexpression of GIPC1 alone in Tgfbr3(-/-) cells did not rescue invasion whereas knockdown of GIPC1 in Tgfbr3(+/+) cells decreased invasion in response to TGFβ2, FGF2, and HMW-HA. We conclude that TGFβR3 interaction with GIPC1 is critical for regulating invasion and growth factor responsiveness in epicardial cells and that dysregulation of epicardial cell proliferation and invasion contributes to failed coronary vessel development in Tgfbr3(-/-) mice.
Copyright © 2011 Elsevier Inc. All rights reserved.
Valvular heart disease is a major cause of mortality and morbidity. Revealing the cellular processes and molecules that regulate valve formation and remodeling is required to develop effective therapies. A key step in valve formation during heart development is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endocardial cells in the atrioventricular cushion (AVC). The type III transforming growth factor-β receptor (TGFβR3) regulates AVC endocardial cell EMT in vitro and mesenchymal cell differentiation in vivo. Little is known concerning the signaling mechanisms downstream of TGFβR3. Here we use endocardial cell EMT in vitro to determine the role of 2 well-characterized downstream TGFβ signaling pathways in TGFβR3-dependent endocardial cell EMT. Targeting of Smad4, the common mediator Smad, demonstrated that Smad signaling is required for EMT in the AVC and TGFβR3-dependent EMT stimulated by TGFβ2 or BMP-2. Although we show that Smads 1, 2, 3, and 5 are required for AVC EMT, overexpression of Smad1 or Smad3 is not sufficient to induce EMT. Consistent with the activation of the Par6/Smurf1 pathway downstream of TGFβR3, targeting ALK5, Par6, or Smurf1 significantly inhibited EMT in response to either TGFβ2 or BMP-2. The requirement for ALK5 activity, Par6, and Smurf1 for TGFβR3-dependent endocardial cell EMT is consistent with the documented role of this pathway in the dissolution of tight junctions. Taken together, our data demonstrate that TGFβR3-dependent endocardial cell EMT stimulated by either TGFβ2 or BMP-2 requires Smad4 and the activation of the Par6/Smurf1 pathway.
Copyright © 2011 S. Karger AG, Basel.
In the developing heart, the epicardium is a major source of progenitor cells that contribute to the formation of the coronary vessel system. These epicardial progenitors give rise to the different cellular components of the coronary vasculature by undergoing a number of morphological and physiological changes collectively known as epithelial to mesenchymal transformation (EMT). However, the specific signaling mechanisms that regulate epicardial EMT are yet to be delineated. In this study we investigated the role of TGFβ2 and hyaluronan (HA) during epicardial EMT and how signals from these two molecules are integrated during this important process. Here we show that TGFβ2 induces MEKK3 activation, which in turn promotes ERK1/2 and ERK5 phosphorylation. TGFβ2 also increases Has2 expression and subsequent HA production. Nevertheless, inhibition of MEKK3 kinase activity, silencing of ERK5 or pharmacological disruption of ERK1/2 activation significantly abrogates this response. Thus, TGFβ2 promotes Has2 expression and HA production through a MEKK3/ERK1/2/5-dependent cascade. Furthermore, TGFβ2 is able to induce epicardial cell invasion and differentiation but not proliferation. However, inhibition of MEKK3-dependent pathways, degradation of HA by hyaluronidases or blockade of CD44, significantly impairs the biological response to TGFβ2. Taken together, these findings demonstrate that TGFβ2 activation of MEKK3/ERK1/2/5 signaling modulates Has2 expression and HA production leading to the induction of EMT events. This is an important and novel mechanism showing how TGFβ2 and HA signals are integrated to regulate changes in epicardial cell behavior.
Published by Elsevier Inc.
The transforming growth factor-beta (TGF-beta) signaling pathway plays dual roles in epithelial cell tumorigenesis. TGF-beta is initially growth inhibitory, but as tumorigenesis progresses, TGF-beta becomes prometastatic. Although the role of the types I and II TGF-beta receptors is fairly well established, the role of the ubiquitously expressed TGF-beta type III receptor (TbetaRIII) in tumorigenesis is less defined. To examine the role of TbetaRIII in breast cancer cells, we stably expressed short hairpin RNAs specific to TbetaRIII in MDA-231 human breast cancer cells and mouse mammary carcinoma cells expressing the polyomavirus middle T oncogene (PMTLuc). MDA-231 and PMTLuc cells with down-regulated TbetaRIII expression (231-kd; PMTLuc-kd) exhibited decreased growth rate, motility, and invasion into Matrigel, as well as an increase in apoptosis, compared with control cells. MDA-231 xenografts established in nude mice metastasized, whereas tumors made by 231-kd cells did not. Nuclear factor-kappaB (NF-kappaB) activity, which is known to regulate cell growth and motility, was lower in the MDA-231 and PMTLuc knockdown cells compared with control cells. Transfection of an expression vector encoding constitutively active IKK2 into the 231-kd cells restored the ability of TbetaRIII-deficient cells to invade Matrigel and decreased their basal level of apoptosis. These data indicate that TbetaRIII differentially regulates cell growth, motility, and invasion in tumorigenic MDA-231 and PMTLuc cells and that these growth changes occur through the modulation of NF-kappaB activity.