Chemical regulators of epithelial plasticity reveal a nuclear receptor pathway controlling myofibroblast differentiation

Plasticity in epithelial tissues relates to processes of embryonic development, tissue fibrosis and cancer progression. Pharmacological modulation of epithelial transitions during disease progression may thus be clinically useful. Using human keratinocytes and a robotic high-content imaging platform, we screened for chemical compounds that reverse transforming growth factor β (TGF-β)-induced epithelial-mesenchymal transition. In addition to TGF-β receptor kinase inhibitors, we identified small molecule epithelial plasticity modulators including a naturally occurring hydroxysterol agonist of the liver X receptors (LXRs), members of the nuclear receptor transcription factor family. Endogenous and synthetic LXR agonists tested in diverse cell models blocked α-smooth muscle actin expression, myofibroblast differentiation and function. Agonist-dependent LXR activity or LXR overexpression in the absence of ligand counteracted TGF-β-mediated myofibroblast terminal differentiation and collagen contraction. The protective effect of LXR agonists against TGF-β-induced pro-fibrotic activity raises the possibility that anti-lipidogenic therapy may be relevant in fibrotic disorders and advanced cancer

Sox17 Promotes Cell Cycle Progression and Inhibits TGF-β/Smad3 Signaling to Initiate Progenitor Cell Behavior in the Respiratory Epithelium

The Sry-related high mobility group box transcription factor Sox17 is required for diverse developmental processes including endoderm formation, vascular development, and fetal hematopoietic stem cell maintenance. Expression of Sox17 in mature respiratory epithelial cells causes proliferation and lineage respecification, suggesting that Sox17 can alter adult lung progenitor cell fate. In this paper, we identify mechanisms by which Sox17 influences lung epithelial progenitor cell behavior and reprograms cell fate in the mature respiratory epithelium. Conditional expression of Sox17 in epithelial cells of the adult mouse lung demonstrated that cell cluster formation and respecification of alveolar progenitor cells toward proximal airway lineages were rapidly reversible processes. Prolonged expression of Sox17 caused the ectopic formation of bronchiolar-like structures with diverse respiratory epithelial cell characteristics in alveolar regions of lung. During initiation of progenitor cell behavior, Sox17 induced proliferation and increased the expression of the progenitor cell marker Sca-1 and genes involved in cell cycle progression. Notably, Sox17 enhanced cyclin D1 expression in vivo and activated cyclin D1 promoter activity in vitro. Sox17 decreased the expression of transforming growth factor-beta (TGF-β)-responsive cell cycle inhibitors in the adult mouse lung, including p15, p21, and p57, and inhibited TGF-β1-mediated transcriptional responses in vitro. Further, Sox17 interacted with Smad3 and blocked Smad3 DNA binding and transcriptional activity. Together, these data show that a subset of mature respiratory epithelial cells retains remarkable phenotypic plasticity and that Sox17, a gene required for early endoderm formation, activates the cell cycle and reinitiates multipotent progenitor cell behavior in mature lung cells

Regulation of endothelial cell plasticity by TGF-β

Recent evidence has demonstrated that endothelial cells can have a remarkable plasticity. By a process called Endothelial-to-Mesenchymal Transition (EndMT) endothelial cells convert to a more mesenchymal cell type that can give rise to cells such as fibroblasts, but also bone cells. EndMT is essential during embryonic development and tissue regeneration. Interestingly, it also plays a role in pathological conditions like fibrosis of organs such as the heart and kidney. In addition, EndMT contributes to the generation of cancer associated fibroblasts that are known to influence the tumor-microenvironment favorable for the tumor cells. EndMT is a form of the more widely known and studied Epithelial-to-Mesenchymal Transition (EMT). Like EMT, EndMT can be induced by transforming growth factor (TGF)-β. Indeed many studies have pointed to the important role of TGF-β receptor/Smad signaling and downstream targets, such as Snail transcriptional repressor in EndMT. By selective targeting of TGF-β receptor signaling pathological EndMT may be inhibited for the therapeutic benefit of patients with cancer and fibrosis

Nomenclature and listing of celiac disease relevant gluten T-cell epitopes restricted by HLA-DQ molecules

Celiac disease is caused by an abnormal intestinal T-cell response to gluten proteins of wheat, barley and rye. Over the last few years, a number of gluten T-cell epitopes restricted by celiac disease associated HLA-DQ molecules have been characterized. In this work, we give an overview of these epitopes and suggest a comprehensive, new nomenclature

CCN2 Is Required for the TGF-β Induced Activation of Smad1 - Erk1/2 Signaling Network

Connective tissue growth factor (CCN2) is a multifunctional matricellular protein, which is frequently overexpressed during organ fibrosis. CCN2 is a mediator of the pro-fibrotic effects of TGF-β in cultured cells, but the specific function of CCN2 in the fibrotic process has not been elucidated. In this study we characterized the CCN2-dependent signaling pathways that are required for the TGF-β induced fibrogenic response. By depleting endogenous CCN2 we show that CCN2 is indispensable for the TGF-β-induced phosphorylation of Smad1 and Erk1/2, but it is unnecessary for the activation of Smad3. TGF-β stimulation triggered formation of the CCN2/β3 integrin protein complexes and activation of Src signaling. Furthermore, we demonstrated that signaling through the αvβ3 integrin receptor and Src was required for the TGF-β induced Smad1 phosphorylation. Recombinant CCN2 activated Src and Erk1/2 signaling, and induced phosphorylation of Fli1, but was unable to stimulate Smad1 or Smad3 phosphorylation. Additional experiments were performed to investigate the role of CCN2 in collagen production. Consistent with the previous studies, blockade of CCN2 abrogated TGF-β-induced collagen mRNA and protein levels. Recombinant CCN2 potently stimulated collagen mRNA levels and upregulated activity of the COL1A2 promoter, however CCN2 was a weak inducer of collagen protein levels. CCN2 stimulation of collagen was dose-dependent with the lower doses (<50 ng/ml) having a stimulatory effect and higher doses having an inhibitory effect on collagen gene expression. In conclusion, our study defines a novel CCN2/αvβ3 integrin/Src/Smad1 axis that contributes to the pro-fibrotic TGF-β signaling and suggests that blockade of this pathway may be beneficial for the treatment of fibrosis

A novel spontaneous model of epithelial-mesenchymal transition (EMT) using a primary prostate cancer derived cell line demonstrating distinct stem-like characteristics

Cells acquire the invasive and migratory properties necessary for the invasion-metastasis cascade and the establishment of aggressive, metastatic disease by reactivating a latent embryonic programme: epithelial-to-mesenchymal transition (EMT). Herein, we report the development of a new, spontaneous model of EMT which involves four phenotypically distinct clones derived from a primary tumour-derived human prostate cancer cell line (OPCT-1), and its use to explore relationships between EMT and the generation of cancer stem cells (CSCs) in prostate cancer. Expression of epithelial (E-cadherin) and mesenchymal markers (vimentin, fibronectin) revealed that two of the four clones were incapable of spontaneously activating EMT, whereas the others contained large populations of EMT-derived, vimentin-positive cells having spindle-like morphology. One of the two EMT-positive clones exhibited aggressive and stem cell-like characteristics, whereas the other was non-aggressive and showed no stem cell phenotype. One of the two EMT-negative clones exhibited aggressive stem cell-like properties, whereas the other was the least aggressive of all clones. These findings demonstrate the existence of distinct, aggressive CSC-like populations in prostate cancer, but, importantly, that not all cells having a potential for EMT exhibit stem cell-like properties. This unique model can be used to further interrogate the biology of EMT in prostate cancer

Estrogen Receptor Alpha Is Expressed in Mesenteric Mesothelial Cells and Is Internalized in Caveolae upon Freund's Adjuvant Treatment

Transformation of epithelial cells into connective tissue cells (epithelial-mesenchymal transition, EMT) is a complex mechanism involved in tumor metastasis, and in normal embryogenesis, while type II EMT is mainly associated with inflammatory events and tissue regenaration. In this study we examined type II EMT at the ultrastructural and molecular level during the inflammatory process induced by Freund's adjuvant treatment in rat mesenteric mesothelial cells. We found that upon the inflammatory stimulus mesothelial cells lost contact with the basal lamina and with each other, and were transformed into spindle-shaped cells. These morphological changes were accompanied by release of interleukins IL-1alpha, -1beta and IL-6 and by secretion of transforming growth factor beta (TGF-beta) into the peritoneal cavity. Mesothelial cells also expressed estrogen receptor alpha (ER-alpha) as shown by immunolabeling at the light and electron microscopical levels, as well as by quantitative RT-PCR. The mRNA level of ER-alpha showed an inverse correlation with the secretion of TGF-beta. At the cellular and subcellular levels ER-alpha was colocalized with the coat protein caveolin-1 and was found in the plasma membrane of mesothelial cells, in caveolae close to multivesicular bodies (MVBs) or in the membrane of these organelles, suggesting that ER-alpha is internalized via caveola-mediated endocytosis during inflammation. We found asymmetric, thickened, electron dense areas on the limiting membrane of MVBs (MVB plaques) indicating that these sites may serve as platforms for collecting and organizing regulatory proteins. Our morphological observations and biochemical data can contribute to form a potential model whereby ER-alpha and its caveola-mediated endocytosis might play role in TGF-beta induced type II EMT in vivo

Loss of expression of TGF-βs and their receptors in chronic skin lesions induced by sulfur mustard as compared with chronic contact dermatitis patients

<p>Abstract</p> <p>Background</p> <p>Sulfur mustard (SM) is a blister-forming agent that has been used as a chemical weapon. Sulfur mustard can cause damage in various organs, especially the skin, respiratory system, and eyes. Generally, the multiple complications of mustard gas result from its alkalizing potency; it reacts with cellular components like DNA, RNA, proteins, and lipid membranes.</p> <p>TGF-β is a multi-functional cytokine with multiple biological effects ranging from cell differentiation and growth inhibition to extracellular matrix stimulation, immunosuppression, and immunomodulation. TGF-β has 3 isoforms (TGF-β 1, 2, 3) and its signaling is mediated by its receptors: R1, R2 and intracellular Smads molecules.</p> <p>TGF-β has been shown to have anti-inflammatory effects. TGF-βs and their receptors also have an important role in modulation of skin inflammation, proliferation of epidermal cells, and wound healing, and they have been implicated in different types of skin inflammatory disorders.</p> <p>Methods</p> <p>Seventeen exposed SM individuals (48.47 ± 9.3 years), 17 chronic dermatitis patients (46.52 ± 14.6 years), and 5 normal controls (44.00 ± 14.6 years) were enrolled in this study.</p> <p>Evaluation of TGF-βs and their receptors expressions was performed by semiquantitative RT-PCR. Only TGF1was analyzed immunohistochemically.</p> <p>Results</p> <p>Our results showed significant decreases in the expression percentages of TGF-β 1, 2 and R1, R2 in chemical victims in comparison with chronic dermatitis and normal subjects and significant decreases in the intensity of R1 and R2 expressions in chemical victims in comparison with chronic dermatitis and normal controls. (P value < 0.05)</p> <p>Conclusions</p> <p>TGF-βs and their receptors appear to have a noticeable role in chronic inflammatory skin lesions caused by sulfur mustard.</p

Dynamic, Large-Scale Profiling of Transcription Factor Activity from Live Cells in 3D Culture

phenotypes. Taken together, our objective was to develop cellular arrays for dynamic, large-scale quantification of TF activity as cells organized into spherical structures within 3D culture.TF-specific and normalization reporter constructs were delivered in parallel to a cellular array containing a well-established breast cancer cell line cultured in Matrigel. Bioluminescence imaging provided a rapid, non-invasive, and sensitive method to quantify luciferase levels, and was applied repeatedly on each sample to monitor dynamic activity. Arrays measuring 28 TFs identified up to 19 active, with 13 factors changing significantly over time. Stimulation of cells with β-estradiol or activin A resulted in differential TF activity profiles evolving from initial stimulation of the ligand. Many TFs changed as expected based on previous reports, yet arrays were able to replicate these results in a single experiment. Additionally, arrays identified TFs that had not previously been linked with activin A.This system provides a method for large-scale, non-invasive, and dynamic quantification of signaling pathway activity as cells organize into structures. The arrays may find utility for investigating mechanisms regulating normal and abnormal tissue growth, biomaterial design, or as a platform for screening therapeutics