The Wnt11 Signaling Pathway in Potential Cellular EMT and Osteochondral Differentiation Progression in Nephrolithiasis Formation

The molecular events leading to nephrolithiasis are extremely complex. Previous studies demonstrated that calcium and transforming growth factor-β1 (TGF-β1) may participate in the pathogenesis of stone formation, but the explicit mechanism has not been defined. Using a self-created genetic hypercalciuric stone-forming (GHS) rat model, we observed that the increased level of serous/uric TGF-β1 and elevated intracellular calcium in primary renal tubular epithelial cells (PRECs) was associated with nephrolithiasis progression in vivo. In the setting of high calcium plus high TGF-β1 in vitro, PRECs showed great potential epithelial to mesenchymal transition (EMT) progression and osteochondral differentiation properties, representing the multifarious increased mesenchymal and osteochondral phenotypes (Zeb1, Snail1, Col2A1, OPN, Sox9, Runx2) and decreased epithelial phenotypes (E-cadherin, CK19) bythe detection of mRNAs and corresponding proteins. Moreover, TGF-β-dependent Wnt11 knockdown and L-type Ca²⁺ channel blocker could greatly reverse EMT progression and osteochondral differentiation in PRECs. TGF-β1 alone could effectively promote EMT, but it had no effect on osteochondral differentiation in NRK cells (Rat kidney epithelial cell line). Stimulation with Ca²⁺ alone did not accelerate differentiation of NRK. Co-incubation of extracellular Ca²⁺ and TGF-β1 synergistically promotes EMT and osteochondral differentiation in NRK control cells. Our data supplied a novel view that the pathogenesis of calcium stone development may be associated with synergic effects of TGF-β1 and Ca²⁺, which promote EMT and osteochondral differentiation via Wnt11 and the L-type calcium channel.     

Loss of the Metastasis Suppressor NME1, But Not of Its Highly Related Isoform NME2, Induces a Hybrid Epithelial–Mesenchymal State in Cancer Cells

Epithelial–mesenchymal transition (EMT) is important for the initial steps of metastasis. Although it is well accepted that the nucleoside diphosphate kinase NME1 is a metastasis suppressor, its effect on EMT remains poorly documented, as does that of its closely related isoform, NME2. Here, by using gene silencing, inactivation and overexpression strategies in a variety of cellular models of cancer, we show that NME1 is a powerful inhibitor of EMT. Genetic manipulation of NME2, by contrast, had no effect on the EMT phenotype of cancer cells, indicating a specific function of NME1 in EMT regulation. Loss of NME1 in epithelial cancer cells resulted in a hybrid phenotype intermediate between epithelial and mesenchymal cells, which is known to be associated with cells with a highly metastatic character. Conversely, overexpression of NME1 in mesenchymal cancer cells resulted in a more epithelial phenotype. We found that NME1 expression was negatively associated with EMT markers in many human cancers and was reduced in human breast tumor cell lines with the aggressive ‘triple-negative’ phenotype when compared to human breast tumor cell lines positive for estrogen receptor. We show that NME1, but not NME2, is an inhibitor of essential concerted intracellular signaling pathways involved in inducing EMT, including the AKT and MAPK (ERK, p38, and JNK) pathways. Additionally, NME1 depletion considerably altered the distribution of E-cadherin, a gatekeeper of the epithelial phenotype, shifting it from the plasma membrane to the cytosol and resulting in less E-cadherin on the cell surface than in control cells. Functional aggregation and dispersion assays demonstrated that inactivation of NME1 decreases E-cadherin-mediated cell–cell adhesion. We conclude that NME1, but not NME2, acts specifically to inhibit EMT and prevent the earliest stages of metastasis.     

Chrysin Ameliorates Cyclosporine-A-Induced Renal Fibrosis by Inhibiting TGF-β1-Induced Epithelial–Mesenchymal Transition

Cyclosporine A (CsA) is a nephrotoxicant that causes fibrosis via induction of epithelial–mesenchymal transition (EMT). The flavonoid chrysin has been reported to have anti-fibrotic activity and inhibit signaling pathways that are activated during EMT. This study investigated the nephroprotective role of chrysin in the prevention of CsA-induced renal fibrosis and elucidated a mechanism of inhibition against CsA-induced EMT in proximal tubule cells. Treatment with chrysin prevented CsA-induced renal dysfunction in Sprague Dawley rats measured by blood urea nitrogen (BUN), serum creatinine and creatinine clearance. Chrysin inhibited CsA-induced tubulointerstitial fibrosis, characterized by reduced tubular damage and collagen deposition. In vitro, chrysin significantly inhibited EMT in LLC-PK₁ cells, evidenced by inhibition of cell migration, decreased collagen expression, reduced presence of mesenchymal markers and elevated epithelial junction proteins. Furthermore, chrysin co-treatment diminished CsA-induced TGF-β₁ signaling pathways, decreasing Smad 3 phosphorylation which lead to a subsequent reduction in Snail expression. Chrysin also inhibited activation of the Akt/ GSK-3β pathway. Inhibition of both pathways diminished the cytosolic accumulation of β-catenin, a known trigger for EMT. In conclusion, flavonoids such as chrysin offer protection against CsA-induced renal dysfunction and interstitial fibrosis. Chrysin was shown to inhibit CsA-induced TGF-β₁-dependent EMT in proximal tubule cells by modulation of Smad-dependent and independent signaling pathways.     

Wnt/β-Catenin Signaling Contributes to Paclitaxel Resistance in Bladder Cancer Cells with Cancer Stem Cell-Like Properties

The Wnt/β-catenin pathway plays an important role in tumor progression and chemotherapy resistance and seems to be essential for the maintenance of cancer stem cells (CSC) in several tumor types. However, the interplay of these factors has not been fully addressed in bladder cancer. Here, our goal was to analyze the role of the Wnt/β-catenin pathway in paclitaxel resistance and to study the therapeutic efficacy of its inhibition in bladder cancer cells, as well as to determine its influence in the maintenance of the CSC-like phenotype in bladder cancer. Our results show that paclitaxel-resistant HT1197 cells have hyperactivation of the Wnt/β-catenin pathway and increased CSC-like properties compared with paclitaxel-sensitive 5637 cells. Paclitaxel sensitivity diminishes in 5637 cells after β-catenin overexpression or when they are grown as tumorspheres, enriched for the CSC-like phenotype. Additionally, downregulation of β-catenin or inhibition with XAV939 sensitizes HT1197 cells to paclitaxel. Moreover, a subset of muscle-invasive bladder carcinomas shows aberrant expression of β-catenin that associates with positive expression of the CSC marker ALDH1A1. In conclusion, we demonstrate that Wnt/β-catenin signaling contributes to paclitaxel resistance in bladder cancer cells with CSC-like properties.     

Epithelial-Cell-Derived Extracellular Vesicles in Pathophysiology of Epithelial Injury and Repair in Chronic Rhinosinusitis: Connecting Immunology in Research Lab to Biomarkers in Clinics

Epithelial barrier disruption and failure of epithelial repair by aberrant epithelial-mesenchymal transition (EMT)-induced basal cells observed in nasal mucosa of chronic rhinosinusitis (CRS) are speculated to play important roles in disease pathophysiology. Microparticles (MPs) are a type of extracellular vesicle (EV) released by budding or shedding from the plasma membrane of activated or apoptotic cells. MPs are detected in nasal lavage fluids (NLFs) and are now receiving attention as potential biomarkers to evaluate the degree of activation of immune cells and injury of structural cells in nasal mucosa of subjects with sinus disease. There are three types of epithelial-cell-derived MPs, which are defined by the expression of different epithelial specific markers on their surface: EpCAM, E-cadherin, and integrin β6 (ITGB6). When these markers are on MPs that are also carrying canonical EMT/mesenchymal markers (Snail (SNAI1); Slug (SNAI2); alpha-smooth muscle actin (αSMA, ACTA2)) or pro- and anti-coagulant molecules (tissue factor (TF); tissue plasminogen activator (tPA); plasminogen activator inhibitor-1 (PAI-1)), they provide insight as to the roles of epithelial activation for EMT or regulation of coagulation in the underlying disease. In this review, we discuss the potential of epithelial MPs as research tools to evaluate status of nasal mucosae of CRS patients in the lab, as well as biomarkers for management and treatment of CRS in the clinic.     

Simulated Microgravity Inhibits Rodent Dermal Fibroblastic Differentiation of Mesenchymal Stem Cells by Suppressing ERK/β-Catenin Signaling Pathway

Studies have shown that bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into dermal fibroblasts to participate in skin-repairing. However, at present, little is known about how microgravity affects dermal fibroblastic differentiation of BMSCs in space. The aim of this study was to investigate the effect of simulated microgravity (SMG) on the differentiation of BMSCs into dermal fibroblasts and the related molecular mechanism. Here, using a 2D-clinostat device to simulate microgravity, we found that SMG inhibited the differentiation and suppressed the Wnt/β-catenin signaling and phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2). After upregulating the Wnt/β-catenin signaling with lithium chloride (LiCl) treatment, we found that the effect of the differentiation was restored. Moreover, the Wnt/β-catenin signaling was upregulated when phosphorylation of ERK1/2 was activated with tert-Butylhydroquinone (tBHQ) treatment. Taken together, our findings suggest that SMG inhibits dermal fibroblastic differentiation of BMSCs by suppressing ERK/β-catenin signaling pathway, inferring that ERK/β-catenin signaling pathway may act as a potential intervention target for repairing skin injury under microgravity conditions.     

Involvement of the ERK/HIF-1α/EMT Pathway in XCL1-Induced Migration of MDA-MB-231 and SK-BR-3 Breast Cancer Cells

Chemokine–receptor interactions play multiple roles in cancer progression. It was reported that the overexpression of X-C motif chemokine receptor 1 (XCR1), a specific receptor for chemokine X-C motif chemokine ligand 1 (XCL1), stimulates the migration of MDA-MB-231 triple-negative breast cancer cells. However, the exact mechanisms of this process remain to be elucidated. Our study found that XCL1 treatment markedly enhanced MDA-MB-231 cell migration. Additionally, XCL1 treatment enhanced epithelial–mesenchymal transition (EMT) of MDA-MB-231 cells via E-cadherin downregulation and upregulation of N-cadherin and vimentin as well as increases in β-catenin nucleus translocation. Furthermore, XCL1 enhanced the expression of hypoxia-inducible factor-1α (HIF-1α) and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Notably, the effects of XCL1 on cell migration and intracellular signaling were negated by knockdown of XCR1 using siRNA, confirming XCR1-mediated actions. Treating MDA-MB-231 cells with U0126, a specific mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, blocked XCL1-induced HIF-1α accumulation and cell migration. The effect of XCL1 on cell migration was also evaluated in ER^-/HER2⁺ SK-BR-3 cells. XCL1 also promoted cell migration, EMT induction, HIF-1α accumulation, and ERK phosphorylation in SK-BR-3 cells. While XCL1 did not exhibit any significant impact on the matrix metalloproteinase (MMP)-2 and -9 expressions in MDA-MB-231 cells, it increased the expression of these enzymes in SK-BR-3 cells. Collectively, our results demonstrate that activation of the ERK/HIF-1α/EMT pathway is involved in the XCL1-induced migration of both MDA-MB-231 and SK-BR-3 breast cancer cells. Based on our findings, the XCL1–XCR1 interaction and its associated signaling molecules may serve as specific targets for the prevention of breast cancer cell migration and metastasis.     

Signaling Involved in Hair Follicle Morphogenesis and Development

Hair follicle morphogenesis depends on Wnt, Shh, Notch, BMP and other signaling pathways interplay between epithelial and mesenchymal cells. The Wnt pathway plays an essential role during hair follicle induction, Shh is involved in morphogenesis and late stage differentiation, Notch signaling determines stem cell fate while BMP is involved in cellular differentiation. The Wnt pathway is considered to be the master regulator during hair follicle morphogenesis. Wnt signaling proceeds through EDA/EDAR/NF-κB signaling. NF-κB regulates the Wnt pathway and acts as a signal mediator by upregulating the expression of Shh ligand. Signal crosstalk between epithelial and mesenchymal cells takes place mainly through primary cilia. Primary cilia formation is initiated with epithelial laminin-511 interaction with dermal β-1 integrin, which also upregulates expression of downstream effectors of Shh pathway in dermal lineage. PDGF signal transduction essential for crosstalk is mediated through epithelial PDGF-A and PDGFRα expressed on the primary cilia. Dermal Shh and PDGF signaling up-regulates dermal noggin expression; noggin is a potent inhibitor of BMP signaling which helps in counteracting BMP mediated β-catenin inhibition. This interplay of signaling between the epithelial and dermal lineage helps in epithelial Shh signal amplification. The dermal Wnt pathway helps in upregulation of epithelial Notch expression. Dysregulation of these pathways leads to certain abnormalities and in some cases even tumor outgrowth.     

Atractylodin Suppresses TGF-β-Mediated Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells and Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is characterized by fibrotic change in alveolar epithelial cells and leads to the irreversible deterioration of pulmonary function. Transforming growth factor-beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in type 2 lung epithelial cells contributes to excessive collagen deposition and plays an important role in IPF. Atractylodin (ATL) is a kind of herbal medicine that has been proven to protect intestinal inflammation and attenuate acute lung injury. Our study aimed to determine whether EMT played a crucial role in the pathogenesis of pulmonary fibrosis and whether EMT can be utilized as a therapeutic target by ATL treatment to mitigate IPF. To address this topic, we took two steps to investigate: 1. Utilization of anin vitro EMT model by treating alveolar epithelial cells (A549 cells) with TGF-β1 followed by ATL treatment for elucidating the underlying pathways, including Smad2/3 hyperphosphorylation, mitogen-activated protein kinase (MAPK) pathway overexpression, Snail and Slug upregulation, and loss of E-cadherin. Utilization of an in vivo lung injury model by treating bleomycin on mice followed by ATL treatment to demonstrate the therapeutic effectiveness, such as, less collagen deposition and lower E-cadherin expression. In conclusion, ATL attenuates TGF-β1-induced EMT in A549 cells and bleomycin-induced pulmonary fibrosis in mice.     

TGF-β Induced CTGF Expression in Human Lung Epithelial Cells through ERK,ADAM17, RSK1, and C/EBPβ Pathways

Background: Lung epithelial cells play critical roles in idiopathic pulmonary fibrosis. Methods: In the present study, we investigated whether transforming growth factor-β (TGF-β)-induced expression of connective tissue growth factor (CTGF) was regulated by the extracellular signal-regulated kinase (ERK)/a disintegrin and metalloproteinase 17 (ADAM17)/ribosomal S6 kinases 1 (RSK1)/CCAAT/enhancer-binding protein β (C/EBPβ) signaling pathway in human lung epithelial cells (A549). Results: Our results revealed that TGF-β-induced CTGF expression was weakened by ADAM17 small interfering RNA (ADAM17 siRNA), TNF-α processing inhibitor-0 (TAPI-0, an ADAM17 inhibitor), U0126 (an ERK inhibitor), RSK1 siRNA, and C/EBPβ siRNA. TGF-β-induced ERK phosphorylation as well as ADAM17 phosphorylation was attenuated by U0126. The TGF-β-induced increase in RSK1 phosphorylation was inhibited by TAPI-0 and U0126. TGF-β-induced C/EBPβ phosphorylation was weakened by U0126, ADAM17 siRNA, and RSK1 siRNA. In addition, TGF-β increased the recruitment of C/EBPβ to the CTGF promoter. Furthermore, TGF-β enhanced fibronectin (FN), an epithelial–mesenchymal transition (EMT) marker, and CTGF mRNA levels and reduced E-cadherin mRNA levels. Moreover, TGF-β-stimulated FN protein expression was reduced by ADAM17 siRNA and CTGF siRNA. Conclusion: The results suggested that TGF-β induces CTGF expression through the ERK/ADAM17/RSK1/C/EBPβ signaling pathway. Moreover, ADAM17 and CTGF participate in TGF-β-induced FN expression in human lung epithelial cells.     

Particulate Matter (PM10) Promotes Cell Invasion through Epithelial–Mesenchymal Transition (EMT) by TGF-β Activation in A549 Lung Cells

Air pollution presents a major environmental problem, inducing harmful effects on human health. Particulate matter of 10 μm or less in diameter (PM₁₀) is considered an important risk factor in lung carcinogenesis. Epithelial–mesenchymal transition (EMT) is a regulatory program capable of inducing invasion and metastasis in cancer. In this study, we demonstrated that PM₁₀ treatment induced phosphorylation of SMAD2/3 and upregulation of SMAD4. We also reported that PM₁₀ increased the expression and protein levels of TGFB1 (TGF-β), as well as EMT markers SNAI1 (Snail), SNAI2 (Slug), ZEB1 (ZEB1), CDH2 (N-cadherin), ACTA2 (α-SMA), and VIM (vimentin) in the lung A549 cell line. Cell exposed to PM₁₀ also showed a decrease in the expression of CDH1 (E-cadherin). We also demonstrated that expression levels of these EMT markers were reduced when cells are transfected with small interfering RNAs (siRNAs) against TGFB1. Interestingly, phosphorylation of SMAD2/3 and upregulation of SMAD induced by PM₁₀ were not affected by transfection of TGFB1 siRNAs. Finally, cells treated with PM₁₀ exhibited an increase in the capacity of invasiveness because of EMT induction. Our results provide new evidence regarding the effect of PM₁₀ in EMT and the acquisition of an invasive phenotype, a hallmark necessary for lung cancer progression.     

Cripto-1 as a Key Factor in Tumor Progression,Epithelial to Mesenchymal Transition and Cancer Stem Cells

Cripto-1 is an essential protein for human development that plays a key role in the early phase of gastrulation in the differentiation of an embryo as well as assists with wound healing processes. Importantly, Cripto-1 induces epithelial to mesenchymal transition to turn fixed epithelial cells into a more mobile mesenchymal phenotype through the downregulation of epithelial adhesion molecules such as E-cadherin, occludins, and claudins, and the upregulation of mesenchymal, mobile proteins, such as N-cadherin, Snail, and Slug. Consequently, Cripto-1’s role in inducing EMT to promote cell motility is beneficial in embryogenesis, but detrimental in the formation, progression and metastasis of malignant tumors. Indeed, Cripto-1 is found to be upregulated in most cancers, such as breast, lung, gastrointestinal, hepatic, renal, cervical, ovarian, prostate, and skin cancers. Through its role in EMT, Cripto-1 can remodel cancer cells to enable them to travel through the extracellular matrix as well as blood and lymphatic vessels to metastasize to different organs. Additionally, Cripto-1 promotes the survival of cancer stem cells, which can lead to relapse in cancer patients.