Analysis of epithelial-mesenchymal transition markers in psoriatic epidermal keratinocytes.
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ABSTRACT:
SUBMITTER: Man XY
PROVIDER: S-EPMC4554915 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background:Keloids are benign fibroproliferative skin lesions that are difficult to treat and become a lifetime predicament for patients. Several treatment modalities have been put forth, but as yet no satisfactory approach to the prevention or treatment of keloids has been identified. The process of epithelial-to-mesenchymal transition (EMT) has been implicated in keloid scarring, as keloid keratinocytes display an EMT-like phenotype. This study investigated the potential of pirfenidone, an antifibrotic agent, to counteract EMT-like alterations in keloid keratinocytes, including gene expression, cell migratory and proliferative functions. Methods:Normal and keloid keratinocytes were isolated from discarded normal skin tissues and from resected keloid tissues, respectively. Cells were quiesced for 24 h without epidermal growth factor DS-Qi1MCDigital and were exposed to transforming growth factor-beta1 (TGF-?1; 10 ng/mL), with or without pirfenidone (400 ?g/mL), for an additional 24 h. The effects of pirfenidone on cytotoxicity, cell migration, cell proliferation, and on expression of genes and proteins involved in EMT were assayed. Statistical significance was determined by two-way ANOVA using Sigma Plot. Results:We found that pirfenidone did not elicit any cytotoxic effect at concentrations up to 1000 ?g/mL. A statistically significant dose-dependent decrease in basal cell proliferation rate was noted in both normal and keloid keratinocytes when exposed to pirfenidone at concentrations ranging from 200 to 1000 ?g/mL. Pirfenidone significantly decreased basal cell migration in both normal and keloid keratinocytes, but a significant decrease in TGF-?1-induced cell migration was seen only in keloid keratinocytes. Significant inhibition of the expression of TGF-?1-induced core EMT genes, namely hyaluronan synthase 2, vimentin, cadherin-11, and wingless-type MMTV integration site family, member 5A along with fibronectin-1, was observed in both normal and keloid keratinocytes treated with pirfenidone. In addition, the protein levels of vimentin and fibronectin were significantly reduced by pirfenidone (400 ?g/mL) in both normal and keloid keratinocytes. Conclusions:For the first time, this study shows the efficacy of pirfenidone in inhibiting the EMT-like phenotype in keratinocytes derived from keloids, suggesting that pirfenidone may counteract a critical contributor of keloid progression and recurrence.
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Project description:The microgravitational environment is known to affect the cellular behaviour inducing modulation of gene expression and enzymatic activities, epigenetic modifications and alterations of the structural organization. Simulated microgravity, obtained in the laboratory setting through the use of a Random Positioning Machine (RPM), represents a well recognized and useful tool for the experimental studies of the cellular adaptations and molecular changes in response to weightlessness. Short exposure of cultured human keratinocytes to the RPM microgravity influences the cellular circadian clock oscillation. Therefore, here we searched for changes on the regenerative ability and response to tissue damage of human epidermal cells through the analysis of the effects of the simulated microgravity on the re-epithelialization phase of the repair and wound healing process. Combining morphological, biochemical and molecular approaches, we found that the simulated microgravity exposure of human keratinocytes promotes a migratory behavior and triggers the epithelial-mesenchymal transition (EMT) through expression of the typical EMT transcription factors and markers, such as Snail1, Snail2 and ZEB2, metalloproteases, mesenchymal adhesion molecules and cytoskeletal components.
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Project description:We have shown that autocrine proliferation of human keratinocytes (KCs) is strongly dependent upon amphiregulin (AREG), whereas blockade of heparin-binding EGF-like growth factor (HB-EGF) inhibits KC migration in scratch wound assays. Here we demonstrate that expression of soluble HB-EGF (sHB-EGF) or full-length transmembrane HB-EGF (proHB-EGF), but not proAREG, results in profound increases in KC migration and invasiveness in monolayer culture. Coincident with these changes, HB-EGF significantly decreases mRNA expression of several epithelial markers including keratins 1, 5, 10, and 14 while increasing expression of markers of cellular motility including SNAI1, ZEB1, COX-2, and MMP1. Immunostaining revealed HB-EGF-induced expression of the mesenchymal protein vimentin and decreased expression of E-cadherin, as well as nuclear translocation of ?-catenin. Suggestive of a trade-off between KC motility and proliferation, overexpression of HB-EGF also reduced KC growth by >90%. We also show that HB-EGF is strongly induced in regenerating epidermis after partial-thickness wounding of human skin. Taken together, our data suggest that expression of HB-EGF in human KCs triggers a migratory and invasive phenotype with many features of epithelial-mesenchymal transition (EMT), which may be beneficial in the context of cutaneous wound healing.
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Project description:Cutaneous squamous cell carcinoma (CSCC) is an epidermal skin cancer that evolves from normal epidermis along several pre-malignant stages. Previously we found specific miRNAs alterations in each step along these stages. miR-199a-3p expression decreases at the transition to later stages. A crucial step for epithelial carcinoma cells to acquire invasive capacity is the disruption of cell-cell contacts and the gain of mesenchymal motile phenotype, a process known as epithelial-to-mesenchymal transition (EMT). This study aims to study the role of decreased expression of miR-199a-3p in keratinocytes' EMT towards carcinogenesis. First, we measured miR-199a-3p in different stages of epidermal carcinogenesis. Then, we applied Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) assay to search for possible biochemical targets of miR-199a-3p and verified that Ras-associated protein B2 (RAP2B) is a bona-fide target of miR-199a-3p. Next, we analyzed RAP2B expression, in CSCC biopsies. Last, we evaluated possible mechanisms leading to decreased miR-199a-3p expression. miR-199a-3p induces a mesenchymal to epithelial transition (MET) in CSSC cells. Many of the under-expressed genes in CSCC overexpressing miR-199a-3p, are possible targets of miR-199a-3p and play roles in EMT. RAP2B is a biochemical target of miR-199a-3p. Overexpression of miR-199a-3p in CSCC results in decreased phosphorylated focal adhesion kinase (FAK). In addition, inhibiting FAK phosphorylation inhibits EMT marker genes' expression. In addition, we proved that DNA methylation is part of the mechanism by which miR-199a-3p expression is inhibited. However, it is not by the methylation of miR-199a putative promoter. These findings suggest that miR-199a-3p inhibits the EMT process by targeting RAP2B. Inhibitors of RAP2B or FAK may be effective therapeutic agents for CSCC.
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Project description:A state of chronic inflammation is common in organs affected by autoimmune disorders, such as autoimmune thyroid diseases (AITD). Epithelial cells, such as thyroid follicular cells (TFCs), can experience a total or partial transition to a mesenchymal phenotype under these conditions. One of the major cytokines involved in this phenomenon is transforming growth factor beta (TGF-β), which, at the initial stages of autoimmune disorders, plays an immunosuppressive role. However, at chronic stages, TGF- β contributes to fibrosis and/or transition to mesenchymal phenotypes. The importance of primary cilia (PC) has grown in recent decades as they have been shown to play a key role in cell signaling and maintaining cell structure and function as mechanoreceptors. Deficiencies of PC can trigger epithelial-mesenchymal transition (EMT) and exacerbate autoimmune diseases. A set of EMT markers (E-cadherin, vimentin, α-SMA, and fibronectin) were evaluated in thyroid tissues from AITD patients and controls through RT-qPCR, immunohistochemistry (IHC), and western blot (WB). We established an in vitro TGF-β-stimulation assay in a human thyroid cell line to assess EMT and PC disruption. EMT markers were evaluated in this model using RT-qPCR and WB, and PC was evaluated with a time-course immunofluorescence assay. We found an increased expression of the mesenchymal markers α-SMA and fibronectin in TFCs in the thyroid glands of AITD patients. Furthermore, E-cadherin expression was maintained in these patients compared to the controls. The TGF-β-stimulation assay showed an increase in EMT markers, including vimentin, α-SMA, and fibronectin in thyroid cells, as well as a disruption of PC. The TFCs from the AITD patients experienced a partial transition to a mesenchymal phenotype, preserving epithelial characteristics associated with a disruption in PC, which might contribute to AITD pathogenesis.
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Project description:In the wound healing process, the morphology of keratinocytes at the wound edge temporarily changes to a spindle morphology, which is thought to occur due to an epithelial-mesenchymal transition (EMT). Fibroblast growth factor (FGF) 2, also called basic FGF, has the potential to accelerate wound closure by activating vascular endothelial cells and fibroblasts. We examined the effects of FGF2 on keratinocyte morphology and EMT in wounded skin. Histological examination of murine wounds treated with FGF2 revealed that wound edge keratinocytes formed thickened and multilayered epithelia. In addition, we detected wound edge keratinocytes migrating individually toward the wound center. These migrating keratinocytes exhibited not only spindle morphology but also down-regulated E-cadherin and up-regulated vimentin expression, which is characteristic of EMT. In FGF2-treated wounds, a PCR array revealed the upregulation of genes related to EMT, including transforming growth factor (TGF) signaling. Further, FGF2-treated wound edge keratinocytes expressed EMT-associated transcription factors, including Snai2, and showed translocation of ?-catenin from the cell membrane to the cytoplasm/nucleus. However, in vitro examination of keratinocytes revealed that FGF2 alone did not activate EMT in keratinocytes, but that FGF2 might promote EMT in combination with TGF?1. These findings suggest that FGF2 treatment of wounds could promote keratinocyte EMT, accelerating wound closure.
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Project description:p63 is a p53 family protein required for morphogenesis and postnatal regeneration of epithelial tissues. Here we demonstrate that ΔNp63α, a p63 isoform lacking the N-terminal transactivation domain, induces epithelial-mesenchymal transition (EMT) in primary human keratinocytes in a TGF-β-dependent manner. Rapidly proliferating normal human epidermal keratinocytes (NHEK) were infected with retroviral vector expressing ΔNp63α or empty vector and serially subcultured until replicative senescence. No phenotypic changes were observed until the culture reached senescence. Then the ΔNp63α-transduced cells underwent morphological changes resembling mesenchymal cells and acquired the EMT phenotype. Treatment with exogenous TGF-β accelerated EMT in presenescent ΔNp63α-transduced cells, whereas the inhibition of TGF-β signaling reversed the EMT phenotype. TGF-β treatment alone led to growth arrest in control NHEK with no evidence of EMT, indicating that ΔNp63α altered the cellular response to TGF-β treatment. ΔNp63α-transduced cells acquiring EMT gained the ability to be differentiated to osteo-/odontogenic and adipogenic pathways, resembling mesenchymal stem cells. Furthermore, these cells expressed enhanced levels of Nanog and Lin28, which are transcription factors associated with pluripotency. These data indicate that EMT required ΔNp63α transduction and intact TGF-β signaling in NHEK.
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Project description:Epithelial-mesenchymal transition (EMT), a crucial event in cancer progression and embryonic development, is induced by transforming growth factor (TGF)-beta in mouse mammary NMuMG epithelial cells. Id proteins have previously been reported to inhibit major features of TGF-beta-induced EMT. In this study, we show that expression of the deltaEF1 family proteins, deltaEF1 (ZEB1) and SIP1, is gradually increased by TGF-beta with expression profiles reciprocal to that of E-cadherin. SIP1 and deltaEF1 each dramatically down-regulated the transcription of E-cadherin in NMuMG cells through direct binding to the E-cadherin promoter. Silencing of the expression of both SIP1 and deltaEF1, but not either alone, completely abolished TGF-beta-induced E-cadherin repression. However, expression of mesenchymal markers, including fibronectin, N-cadherin, and vimentin, was not affected by knockdown of SIP1 and deltaEF1. TGF-beta-induced the expression of Ets1, which in turn activated deltaEF1 promoter activity. Moreover, up-regulation of SIP1 and deltaEF1 expression by TGF-beta was suppressed by knockdown of Ets1 expression. In addition, Id2 suppressed the TGF-beta- and Ets1-induced up-regulation of deltaEF1. Taken together, these findings suggest that the deltaEF1 family proteins, SIP1 and deltaEF1, are necessary, but not sufficient, for TGF-beta-induced EMT and that Ets1 induced by TGF-beta may function as an upstream transcriptional regulator of SIP1 and deltaEF1.
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Project description:The epithelial to mensenchymal transition program regulates various aspects of embryonic development and tissue homeostasis, but aberrant activation of this pathway in cancer contributes to tumor progression and metastasis. TGF-b potently induces an epithelial to mensenchymal transition in cancers of epithelial origin by inducing transcriptional changes mediated by several key transcription factors. Here, we identify the developmental transcription factor SOX4 as a transcriptional target of TGF-b in immortalized human mammary epithelial cells. SOX4 expression and activity are rapidly induced in the early stages of the TGF-b-induced epithelial to mensenchymal transition. We demonstrate that conditional activation of Sox4 is sufficient to induce the expression of N-cadherin and additional mesenchymal markers including vimentin and fibronectin, but fails to induce complete EMT as no changes are observed in the expression of E-cadherin and b-catenin. Moreover, shRNA-mediated knockdown of SOX4 significantly delays TGF-b-induced mRNA and protein expression of mesenchymal markers. Taken together, these data suggest that TGF-b-mediated increased expression of SOX4 is required for the induction of a mesenchymal phenotype during EMT in human mammary epithelial cells.
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Project description:Acute gastroenteritis (AGE) is a serious global health problem and has been known to cause millions of infant deaths every year. Rotavirus (RV), a member of the Reoviridae family, still majorly accounts for the AGE in children below 5 years of age in India and worldwide. The involvement of miRNAs in the pathogenesis of RV has been suggested to be of the proviral as well as the anti-viral nature. miRNAs that promote the RV pathogenesis are capable of targeting the cellular components to evade the host anti-viral strategies. On the other hand, miRNAs with anti-rotaviral properties are themselves incapacitated during the progression of the infection. The exploitation of the epithelial-mesenchymal transition (EMT) as a pro-rotaviral strategy has already been identified. Thus, miRNAs that proficiently target the intermediates of the EMT pathway may serve as anti-viral counterparts in the RV-host interactions. The role of microRNA-29b (miR-29b) in the majority of human cancers has been well demonstrated, but its significance in viral infections is yet to be elaborated. In this study, we have assessed the role of miR-29b in RV-induced EMT and RV replication. Our study on miR-29b provides evidence for the recruitment of RV non-structural protein NSP1 to control the trans-repression of miR-29b in a p53-dependent manner. The trans-repression of miR-29b modulates the EMT pathway by targeting tripartite motif-containing protein 44 (TRIM44) and cyclin E1 (CCNE1). SLUG and SNAIL transcription repressors (downstream of TRIM44 and CCNE1) regulate the expression of E-cadherin, an important marker of the EMT. Also, it is established that ectopic expression of miR-29b not only constrains the EMT pathway but also restricts RV replication. Therefore, miR-29b repression is a crucial event in the RV pathogenesis. Ectopic expression of miR-29b displays potential anti-viral properties against RV propagation.
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