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-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-induced EMT transition was analyzed by RNA-Seq in the absence or presence of inducible shRNA-mediated silencing of RELA. In WT cells, TGFβ stimulation significantly affected the expression of 2,441 genes. Gene set enrichment analysis identified WNT, cadherin, and NF-κB signaling as the most prominent TGFβ-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/β-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 TGFβ-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.
Highlights
The epithelial-mesenchymal transition (EMT) is a multistep dedifferentiation program important in tissue repair
We previously characterized a reproducible model of type II EMT using a continuously replicating line of human small airway epithelial cells (hSAECs) immortalized with human telomerase [6, 17]
We found a pattern of basal and transforming growth factor (TGF)-increased RELA binding on the JUN promoter similar to that observed for SNAI1 in XChIP (Fig. 8D)
Summary
The NFB subunit RELA is a master transcriptional regulator of the committed epithelial-mesenchymal transition in airway epithelial cells. Gene expression patterns of SMAD are controlled in a cell type–specific manner by interaction with master transcription factors that function in coordinately regulated complexes to form acetylated histone-rich superenhancers on mesenchymal genes, promoting chromatin remodeling [8, 9]. Time-resolved RNA profiling coupled with ChIP-sequencing studies have elucidated the role of the JUNB complex as an example of a “master” transcription factor of the mesenchymal state [9] Another master regulator is zinc finger E-box– binding homeobox 1 (ZEB1), a bifunctional transcriptional regulator activated by SNAI1 that down-regulates E-cadherin and the EMT-suppressing microRNA-200 family in a complex with the C-terminal binding protein repressor (10 –12). The role of RELA in the initiation of type II EMT has not been determined To address this gap, we conducted time course experiments during the initiation of TGF-induced mesenchymal transition in primary human small airway epithelial cells (hSAECs). These data implicate RELA as a master transcription regulator of type II EMT
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