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The NF?B subunit RELA is a master transcriptional regulator of the committed epithelial-mesenchymal transition in airway epithelial cells.

ABSTRACT: The epithelial-mesenchymal transition (EMT) is a multistep dedifferentiation program important in tissue repair. Here, we examined the role of the transcriptional regulator NF-?B in EMT of primary human small airway epithelial cells (hSAECs). Surprisingly, transforming growth factor ? (TGF?) activated NF-?B/RELA proto-oncogene, NF-?B subunit (RELA) translocation within 1 day of stimulation, yet induction of its downstream gene regulatory network occurred only after 3 days. A time course of TGF?-induced EMT transition was analyzed by RNA-Seq in the absence or presence of inducible shRNA-mediated silencing of RELA. In WT cells, TGF? stimulation significantly affected the expression of 2,441 genes. Gene set enrichment analysis identified WNT, cadherin, and NF-?B signaling as the most prominent TGF?-inducible pathways. By comparison, RELA controlled expression of 3,138 overlapping genes mapping to WNT, cadherin, and chemokine signaling pathways. Conducting upstream regulator analysis, we found that RELA controls six clusters of upstream transcription factors, many of which overlapped with a transcription factor topology map of EMT developed earlier. RELA triggered expression of three key EMT pathways: 1) the WNT/?-catenin morphogen pathway, 2) the JUN transcription factor, and 3) the Snail family transcriptional repressor 1 (SNAI1). RELA binding to target genes was confirmed by ChIP. Experiments independently validating WNT dependence on RELA were performed by silencing RELA via genome editing and indicated that TGF?-induced WNT5B expression and downstream activation of the WNT target AXIN2 are RELA-dependent. We conclude that RELA is a master transcriptional regulator of EMT upstream of WNT morphogen, JUN, SNAI1-ZEB1, and interleukin-6 autocrine loops.

SUBMITTER: Tian B

PROVIDER: S-EPMC6200927 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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The NFκB subunit RELA is a master transcriptional regulator of the committed epithelial-mesenchymal transition in airway epithelial cells.

Tian Bing B Widen Steven G SG Yang Jun J Wood Thomas G TG Kudlicki Andrzej A Zhao Yingxin Y Brasier Allan R AR

The Journal of biological chemistry 20180830 42

The epithelial-mesenchymal transition (EMT) is a multistep dedifferentiation program important in tissue repair. Here, we examined the role of the transcriptional regulator NF-κB in EMT of primary human small airway epithelial cells (hSAECs). Surprisingly, transforming growth factor β (TGFβ) activated NF-κB/RELA proto-oncogene, NF-κB subunit (RELA) translocation within 1 day of stimulation, yet induction of its downstream gene regulatory network occurred only after 3 days. A time course of TGFβ- ...[more]

PMID: 30166344

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Project description:STUDY QUESTION:Does the upregulation of the zinc finger E-box binding homeobox 2 (ZEB2) transcription factor in human trophoblast cells lead to alterations in gene expression consistent with an epithelial-mesenchymal transition (EMT) and a consequent increase in invasiveness? SUMMARY ANSWER:Overexpression of ZEB2 results in an epithelial-mesenchymal shift in gene expression accompanied by a substantial increase in the invasive capacity of human trophoblast cells. WHAT IS KNOWN ALREADY:In-vivo results have shown that cytotrophoblast differentiation into extravillous trophoblast involves an epithelial-mesenchymal transition. The only EMT master regulatory factor which shows changes consistent with extravillous trophoblast EMT status and invasive capacity is the ZEB2 transcription factor. STUDY DESIGN, SIZE, DURATION:This study is a mechanistic investigation of the role of ZEB2 in trophoblast differentiation. We generated stable ZEB2 overexpression clones using the epithelial BeWo and JEG3 choriocarcinoma lines. Using these clones, we investigated the effects of ZEB2 overexpression on the expression of EMT-associated genes and proteins, cell morphology and invasive capability. PARTICIPANTS/MATERIALS, SETTING, METHODS:We used lentiviral transduction to overexpress ZEB2 in BeWo and JEG3 cells. Stable clones were selected based on ZEB2 expression and morphology. A PCR array of EMT-associated genes was used to probe gene expression. Protein measurements were performed by western blotting. Gain-of-function was assessed by quantitatively measuring cell invasion rates using a Transwell assay, a 3D bioprinted placenta model and the xCelligenceTM platform. MAIN RESULTS AND THE ROLE OF CHANCE:The four selected clones (2 × BeWo, 2 × JEG3, based on ZEB2 expression and morphology) all showed gene expression changes indicative of an EMT. The two clones (1 × BeWo, 1 × JEG3) showing >40-fold increase in ZEB2 expression also displayed increased ZEB2 protein; the others, with increases in ZEB2 expression <14-fold did not. The two high ZEB2-expressing clones demonstrated robust increases in invasive capacity, as assessed by three types of invasion assay. These data identify ZEB2-mediated transcription as a key mechanism transforming the epithelial-like trophoblast into cells with a mesenchymal, invasive phenotype. LARGE SCALE DATA:PCR array data have been deposited in the GEO database under accession number GSE116532. LIMITATIONS, REASONS FOR CAUTION:These are in-vitro studies using choriocarcinoma cells and so the results should be interpreted in view of these limitations. Nevertheless, the data are consistent with in-vivo findings and are replicated in two different cell lines. WIDER IMPLICATIONS OF THE FINDINGS:The combination of these data with the in-vivo findings clearly identify ZEB2-mediated EMT as the mechanism for cytotrophoblast differentiation into extravillous trophoblast. Having characterized these cellular mechanisms, it will now be possible to identify the intracellular and extracellular regulatory components which control ZEB2 and trophoblast differentiation. It will also be possible to identify the aberrant factors which alter differentiation in invasive pathologies such as preeclampsia and abnormally invasive placenta (AKA accreta, increta, percreta). STUDY FUNDING AND COMPETING INTEREST(s):Funding was provided by the Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine and Surgery at Hackensack Meridian Health, Hackensack, NJ. The 3D bioprinted placental model work done in Drs Kim and Fisher's labs was supported by the Children's National Medical Center. The xCELLigence work done in Dr Birge's lab was supported by NIH CA165077. The authors declare no competing interests.

Project description:The airway epithelial cell plays a central role in coordinating the pulmonary response to injury and inflammation. Here, transforming growth factor-? (TGF?) activates gene expression programs to induce stem cell-like properties, inhibit expression of differentiated epithelial adhesion proteins and express mesenchymal contractile proteins. This process is known as epithelial mesenchymal transition (EMT); although much is known about the role of EMT in cellular metastasis in an oncogene-transformed cell, less is known about Type II EMT, that occurring in normal epithelial cells. In this study, we applied next generation sequencing (RNA-Seq) in primary human airway epithelial cells to understand the gene program controlling Type II EMT and how cytokine-induced inflammation modifies it.Generalized linear modeling was performed on a two-factor RNA-Seq experiment of 6 treatments of telomerase immortalized human small airway epithelial cells (3 replicates). Using a stringent cut-off, we identified 3,478 differentially expressed genes (DEGs) in response to EMT. Unbiased transcription factor enrichment analysis identified three clusters of EMT regulators, one including SMADs/TP63 and another NF-?B/RelA. Surprisingly, we also observed 527 of the EMT DEGs were also regulated by the TNF-NF-?B/RelA pathway. This Type II EMT program was compared to Type III EMT in TGF? stimulated A549 alveolar lung cancer cells, revealing significant functional differences. Moreover, we observe that Type II EMT modifies the outcome of the TNF program, reducing IFN signaling and enhancing integrin signaling. We confirmed experimentally that TGF?-induced the NF-?B/RelA pathway by observing a 2-fold change in NF-?B/RelA nuclear translocation. A small molecule IKK inhibitor blocked TGF?-induced core transcription factor (SNAIL1, ZEB1 and Twist1) and mesenchymal gene (FN1 and VIM) expression.These data indicate that NF-?B/RelA controls a SMAD-independent gene network whose regulation is required for initiation of Type II EMT. Type II EMT dramatically affects the induction and kinetics of TNF-dependent gene networks.

Project description:The airway epithelial cell plays a central role in coordinating pulmonary response to injury and inflammation. Here, transforming growth factor-b (TGFb) activates gene expression programs to induce stem cell-like properties, inhibit expression of differentiated epithelial adhesion proteins and express mesenchymal contractile proteins. This process is known as epithelial mesenchymal transition (EMT); although much is known about the role of EMT in cellular metastasis in an oncogene-transformed cell, less is known about Type II EMT, that occurring in normal epithelial cells. In this study, we applied next generation sequencing (RNA-seq) in primary human airway epithelial cells to understand the gene program controlling Type II EMT and how cytokine-induced inflammation modifies it. Generalized linear modeling was performed on a two-factor RNA-seq experiment of 6 treatments of telomerase immortalized human small airway epithelial cells (3 replicates). Using a stringent cut-off, we identified 3,478 differentially expressed genes (DEGs) in response to EMT. Unbiased transcription factor enrichment analysis identified three clusters of EMT regulators, one including SMADs/TP63 and another NF-kB/RelA. Surprisingly, we also observed 527 of the EMT DEGs were also regulated by the TNF-NF-kB/RelA pathway. This Type II EMT program was compared to Type III EMT in TGFb stimulated A549 alveolar lung cancer cells, revealing significant functional differences. Moreover, we observe that Type II EMT modifies the outcome of the TNF program, reducing IFN signaling and enhancing integrin signaling. We confirmed experimentally that TGFb-induced the NF-kB/RelA pathway by observing a 2-fold change in NF-kB/RelA nuclear translocation. A small molecule IKK inhibitor blocked TGFb-induced core transcription factor (SNAIL1, ZEB1 and Twist1) and mesenchymal gene (FN1 and VIM) expression. These data indicate that NF-kB/RelA controls a SMAD-independent gene network whose regulation is required for initiation of Type II EMT. Type II EMT dramatically affects the induction and kinetics of TNF-dependent gene networks.

Dataset Information

The NF?B subunit RELA is a master transcriptional regulator of the committed epithelial-mesenchymal transition in airway epithelial cells.

Publications

The NFκB subunit RELA is a master transcriptional regulator of the committed epithelial-mesenchymal transition in airway epithelial cells.

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