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1. Crosstalk between ERK and MRTF‐A signaling regulates TGFβ1‐induced epithelial‐mesenchymal transition

   https://doi.org/10.1002/jcp.30705  

   Nalluri, Sandeep M.; Sankhe, Chinmay S.; O'Connor, Joseph W.; Blanchard, Paul L.; Khouri, Joelle N.; Phan, Steven H.; Virgi, Gage; Gomez, Esther W. (February 2022, Journal of Cellular Physiology)

   Abstract Epithelial‐mesenchymal transition (EMT) is a physiological process that is essential during embryogenesis and wound healing and also contributes to pathologies including fibrosis and cancer. EMT is characterized by marked gene expression changes, loss of cell–cell contacts, remodeling of the cytoskeleton, and acquisition of enhanced motility. In the late stages of EMT, cells can exhibit myofibroblast‐like properties with enhanced expression of the mesenchymal protein marker α‐smooth muscle actin and contractile activity. Transforming growth factor (TGF)‐β1 is a well‐known inducer of EMT and it activates a plethora of signaling cascades including extracellular signal‐regulated kinase (ERK). Previous reports have demonstrated a role for ERK signaling in the early stages of EMT, but the molecular impacts of ERK signaling on the late stages of EMT are still unknown. Here, we found that inhibition of the phosphorylation of ERK enhances focal adhesions, stress fiber formation, cell contractility, and gene expression changes associated with TGFβ1‐induced EMT in mammary epithelial cells. These effects are mediated in part by the phosphorylation state and subcellular localization of myocardin‐related transcription factor‐A. These findings indicate that the intricate crosstalk between signaling cascades plays an important role in regulating the progression of EMT and suggests new approaches to control EMT processes. 

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2. Extracellular matrix viscoelasticity regulates TGFβ1‐induced epithelial‐mesenchymal transition and apoptosis via integrin linked kinase

   https://doi.org/10.1002/jcp.31165  

   Sacco, Jessica L.; Vaneman, Zachary T.; Gomez, Esther W. (December 2023, Journal of Cellular Physiology)

   Abstract Transforming growth factor (TGF)‐β1 is a multifunctional cytokine that plays important roles in health and disease. Previous studies have revealed that TGFβ1 activation, signaling, and downstream cell responses including epithelial‐mesenchymal transition (EMT) and apoptosis are regulated by the elasticity or stiffness of the extracellular matrix. However, tissues within the body are not purely elastic, rather they are viscoelastic. How matrix viscoelasticity impacts cell fate decisions downstream of TGFβ1 remains unknown. Here, we synthesized polyacrylamide hydrogels that mimic the viscoelastic properties of breast tumor tissue. We found that increasing matrix viscous dissipation reduces TGFβ1‐induced cell spreading, F‐actin stress fiber formation, and EMT‐associated gene expression changes, and promotes TGFβ1‐induced apoptosis in mammary epithelial cells. Furthermore, TGFβ1‐induced expression of integrin linked kinase (ILK) and colocalization of ILK with vinculin at cell adhesions is attenuated in mammary epithelial cells cultured on viscoelastic substrata in comparison to cells cultured on nearly elastic substrata. Overexpression of ILK promotes TGFβ1‐induced EMT and reduces apoptosis in cells cultured on viscoelastic substrata, suggesting that ILK plays an important role in regulating cell fate downstream of TGFβ1 in response to matrix viscoelasticity. 

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3. Identification of EMT signaling cross-talk and gene regulatory networks by single-cell RNA sequencing

   https://doi.org/10.1073/pnas.2102050118  

   Deshmukh, Abhijeet P.; Vasaikar, Suhas V.; Tomczak, Katarzyna; Tripathi, Shubham; den Hollander, Petra; Arslan, Emre; Chakraborty, Priyanka; Soundararajan, Rama; Jolly, Mohit Kumar; Rai, Kunal; et al (May 2021, Proceedings of the National Academy of Sciences)

   The epithelial-to-mesenchymal transition (EMT) plays a critical role during normal development and in cancer progression. EMT is induced by various signaling pathways, including TGF-β, BMP, Wnt–β-catenin, NOTCH, Shh, and receptor tyrosine kinases. In this study, we performed single-cell RNA sequencing on MCF10A cells undergoing EMT by TGF-β1 stimulation. Our comprehensive analysis revealed that cells progress through EMT at different paces. Using pseudotime clustering reconstruction of gene-expression profiles during EMT, we found sequential and parallel activation of EMT signaling pathways. We also observed various transitional cellular states during EMT. We identified regulatory signaling nodes that drive EMT with the expression of important microRNAs and transcription factors. Using a random circuit perturbation methodology, we demonstrate that the NOTCH signaling pathway acts as a key driver of TGF-β–induced EMT. Furthermore, we demonstrate that the gene signatures of pseudotime clusters corresponding to the intermediate hybrid EMT state are associated with poor patient outcome. Overall, this study provides insight into context-specific drivers of cancer progression and highlights the complexities of the EMT process. 

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4. Shear stress magnitude and transforming growth factor-βeta 1 regulate endothelial to mesenchymal transformation in a three-dimensional culture microfluidic device

   https://doi.org/10.1039/C6RA16607E  

   Mina, Sara G.; Wang, Wei; Cao, Qingfeng; Huang, Peter; Murray, Bruce T.; Mahler, Gretchen J. (January 2016, RSC Advances)

   Normal fibroblasts are present within the extracellular matrix (ECM). They can become activated, leading to increased proliferation and ECM protein secretion such as collagen type I to promote tissue remodeling. These cells are also involved in adult pathologies including cancer metastasis and cardiac and renal fibrosis. One source of activated fibroblasts is endothelial to mesenchymal transformation (EndMT), in which endothelial cells lose their cell–cell and cell–ECM adhesions, gain invasive properties, and become mesenchymal cells. While EndMT is well characterized in developmental biology, the mechanisms and functional role of EndMT in adult physiology and pathology have not been fully investigated. A microfluidic device with an incorporated three-dimensional ECM chamber was developed to study the role of combined steady fluid shear stress magnitudes and transforming growth factor-βeta 1 (TGF-β1) on EndMT. Low (1 dyne per cm 2 ) steady shear stress and TGF-β1 exposure induced EndMT in endothelial cells, including upregulation of mesenchymal protein and gene expression markers. Cells exposed to TGF-β1 and high (20 dynes per cm 2 ) steady shear stress did not undergo EndMT, and protein and gene expression of mesenchymal markers was significantly downregulated. Mesenchymally transformed cells under static conditions with and without TGF-β1 showed significantly more collagen production when compared to fluidic conditions. These results confirm that both low shear stress and TGF-β1 induce EndMT in endothelial cells, but this process can be prevented by exposure to physiologically relevant high shear stress. These results also show conditions most likely to cause tissue pathology. 

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5. Modulated Fibrosis and Mechanosensing of Fibroblasts by SB525334 in Pediatric Subglottic Stenosis

   https://doi.org/10.1002/lary.30873  

   Ali Akbari Ghavimi, Soheila; Aronson, Matthew R.; Ghaderi, Daniel D.; Friedman, Ryan M.; Patel, Neil; Giordano, Terri; Borek, Ryan C.; Devine, Conor M.; Han, Lin; Jacobs, Ian N.; et al (July 2023, The Laryngoscope)

   ObjectiveSubglottic stenosis (SGS) may result from prolonged intubation where fibrotic scar tissue narrows the airway. The scar forms by differentiated myofibroblasts secreting excessive extracellular matrix (ECM). TGF‐β1 is widely accepted as a regulator of fibrosis; however, it is unclear how biomechanical pathways co‐regulate fibrosis. Therefore, we phenotyped fibroblasts from pediatric patients with SGS to explore how key signaling pathways, TGF‐β and Hippo, impact scarring and assess the impact of inhibiting these pathways with potential therapeutic small molecules SB525334 and DRD1 agonist dihydrexidine hydrochloride (DHX). MethodsLaryngeal fibroblasts isolated from subglottic as well as distal control biopsies of patients with evolving and maturing subglottic stenosis were assessed by α‐smooth muscle actin immunostaining and gene expression for α‐SMA, FN, HGF, and CTGF markers. TGF‐β and Hippo signaling pathways were modulated during TGF‐β1‐induced fibrosis using the inhibitor SB525334 or DHX and analyzed by RT‐qPCR for differential gene expression and atomic force microscopy for ECM stiffness. ResultsSGS fibroblasts exhibited higher α‐SMA staining and greater inflammatory cytokine and fibrotic marker expression upon TGF‐β1 stimulation (p < 0.05). SB525334 restored levels to baseline by reducing SMAD2/3 nuclear translocation (p < 0.0001) and pro‐fibrotic gene expression (p < 0.05). ECM stiffness of stenotic fibroblasts was greater than healthy fibroblasts and was restored to baseline by Hippo pathway modulation using SB525334 and DHX (p < 0.01). ConclusionWe demonstrate that distinct fibroblast phenotypes from diseased and healthy regions of pediatric SGS patients respond differently to TGF‐β1 stimulation, and SB525334 has the superior potential for subglottic stenosis treatment by simultaneously modulating TGF‐β and Hippo signaling pathways. Level of EvidenceNALaryngoscope, 134:287–296, 2024 

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