Regulation of CXCL1 chemokine and CSF3 cytokine levels in myometrial cells by the MAFF transcription factor.

Cytokines play key roles in a variety of reproductive processes including normal parturition as well as preterm birth. Our previous data have shown that MAFF, a member of the MAF family of bZIP transcription factors, is rapidly induced by pro-inflammatory cytokines in PHM1-31 myometrial cells. We performed loss-of-function studies in PHM1-31 cells to identify MAFF dependent genes. We showed that knockdown of MAFF significantly decreased CXCL1 chemokine and CSF3 cytokine transcript and protein levels. Using chromatin immunoprecipitation analyzes, we confirmed CXCL1 and CSF3 genes as direct MAFF targets. We also demonstrated that MAFF function in PHM1-31 myometrial cells is able to control cytokine and matrix metalloproteinase gene expression in THP-1 monocytic cells in a paracrine fashion. Our studies provide valuable insights into the MAFF dependent transcriptional network governing myometrial cell function. The data suggest a role of MAFF in parturition and/or infection-induced preterm labour through modulation of inflammatory processes in the microenvironment.

Highlights of glycosylation and adhesion related genes involved in myogenesis.

BACKGROUND: Myogenesis is initiated by myoblast differentiation and fusion to form myotubes and muscle fibres. A population of myoblasts, known as satellite cells, is responsible for post-natal growth of muscle and for its regeneration. This differentiation requires many changes in cell behaviour and its surrounding environment. These modifications are tightly regulated over time and can be characterized through the study of changes in gene expression associated with this process. During the initial myogenesis steps, using the myoblast cell line C2C12 as a model, Janot et al. (2009) showed significant variations in expression for genes involved in pathways of glycolipid synthesis. In this study we used murine satellite cells (MSC) and their ability to differentiate into myotubes or early fat storage cells to select glycosylation related genes whose variation of expression is myogenesis specific. RESULTS: The comparison of variant genes in both MSC differentiation pathways identified 67 genes associated with myogenesis. Comparison with data obtained for C2C12 revealed that only 14 genes had similar expression profiles in both cell types and that 17 genes were specifically regulated in MSC. Results were validated statistically by without a priori clustering. Classification according to protein function encoded by these 31 genes showed that the main regulated cellular processes during this differentiation were (i) remodeling of the extracellular matrix, particularly, sulfated structures, (ii) down-regulation of O-mannosyl glycan biosynthesis, and (iii) an increase in adhesion protein expression. A functional study was performed on Itga11 and Chst5 encoding two highly up-regulated proteins. The inactivation of Chst5 by specific shRNA delayed the fusion of MSC. By contrast, the inactivation of Itga11 by specific shRNA dramatically decreased the fusion ability of MSC. This result was confirmed by neutralization of Itga11 product by specific antibodies. CONCLUSIONS: Our screening method detected 31 genes specific for myogenic differentiation out of the 383 genes studied. According to their function, interaction networks of the products of these selected genes converged to cell fusion. Functional studies on Itga11 and Chst5 demonstrated the robustness of this screening.

NRF3 suppresses squamous carcinogenesis, involving the unfolded protein response regulator HSPA5.

Epithelial skin cancers are extremely common, but the mechanisms underlying their malignant progression are still poorly defined. Here, we identify the NRF3 transcription factor as a tumor suppressor in the skin. NRF3 protein expression is strongly downregulated or even absent in invasively growing cancer cells of patients with basal and squamous cell carcinomas (BCC and SCC). NRF3 deficiency promoted malignant conversion of chemically induced skin tumors in immunocompetent mice, clonogenic growth and migration of human SCC cells, their invasiveness in 3D cultures, and xenograft tumor formation. Mechanistically, the tumor-suppressive effect of NRF3 involves HSPA5, a key regulator of the unfolded protein response, which we identified as a potential NRF3 interactor. HSPA5 levels increased in the absence of NRF3, thereby promoting cancer cell survival and migration. Pharmacological inhibition or knock-down of HSPA5 rescued the malignant features of NRF3-deficient SCC cells in vitro and in preclinical mouse models. Together with the strong expression of HSPA5 in NRF3-deficient cancer cells of SCC patients, these results suggest HSPA5 inhibition as a treatment strategy for these malignancies in stratified cancer patients.

Loss of NFE2L3 protects against inflammation-induced colorectal cancer through modulation of the tumor microenvironment.

We investigated the role of the NFE2L3 transcription factor in inflammation-induced colorectal cancer. Our studies revealed that Nfe2l3-/- mice exhibit significantly less inflammation in the colon, reduced tumor size and numbers, and skewed localization of tumors with a more pronounced decrease of tumors in the distal colon. CIBERSORT analysis of RNA-seq data from normal and tumor tissue predicted a reduction in mast cells in Nfe2l3-/- animals, which was confirmed by toluidine blue staining. Concomitantly, the transcript levels of Il33 and Rab27a, both important regulators of mast cells, were reduced and increased, respectively, in the colorectal tumors of Nfe2l3-/- mice. Furthermore, we validated NFE2L3 binding to the regulatory sequences of the IL33 and RAB27A loci in human colorectal carcinoma cells. Using digital spatial profiling, we found that Nfe2l3-/- mice presented elevated FOXP3 and immune checkpoint markers CTLA4, TIM3, and LAG3, suggesting an increase in Treg counts. Staining for CD3 and FOXP3 confirmed a significant increase in immunosuppressive Tregs in the colon of Nfe2l3-/- animals. Also, Human Microbiome Project (HMP2) data showed that NFE2L3 transcript levels are higher in the rectum of ulcerative colitis patients. The observed changes in the tumor microenvironment provide new insights into the molecular differences regarding colon cancer sidedness. This may be exploited for the treatment of early-onset colorectal cancer as this emerging subtype primarily displays distal/left-sided tumors.

Profound downregulation of neural transcription factor Npas4 and Nr4a family in fetal mice neurons infected with Zika virus.

Zika virus (ZIKV) infection of neurons leads to neurological complications and congenital malformations of the brain of neonates. To date, ZIKV mechanism of infection and pathogenesis is not entirely understood and different studies on gene regulation of ZIKV-infected cells have identified a dysregulation of inflammatory and stem cell maintenance pathways. MicroRNAs (miRNAs) are post-transcriptional regulators of cellular genes and they contribute to cell development in normal function and disease. Previous reports with integrative analyses of messenger RNAs (mRNAs) and miRNAs during ZIKV infection have not identified neurological pathway defects. We hypothesized that dysregulation of pathways involved in neurological functions will be identified by RNA profiling of ZIKV-infected fetal neurons. We therefore used microarrays to analyze gene expression levels following ZIKV infection of fetal murine neurons. We observed that the expression levels of transcription factors such as neural PAS domain protein 4 (Npas4) and of three members of the orphan nuclear receptor 4 (Nr4a) were severely decreased after viral infection. We confirmed that their downregulation was at both the mRNA level and at the protein level. The dysregulation of these transcription factors has been previously linked to aberrant neural functions and development. We next examined the miRNA expression profile in infected primary murine neurons by microarray and found that various miRNAs were dysregulated upon ZIKV infection. An integrative analysis of the differentially expressed miRNAs and mRNAs indicated that miR-7013-5p targets Nr4a3 gene. Using miRmimics, we corroborated that miR-7013-5p downregulates Nr4a3 mRNA and protein levels. Our data identify a profound dysregulation of neural transcription factors with an overexpression of miR-7013-5p that results in decreased Nr4a3 expression, likely a main contributor to ZIKV-induced neuronal dysfunction.

NFE2L3 Controls Colon Cancer Cell Growth through Regulation of DUX4, a CDK1 Inhibitor.

Constitutive nuclear factor κB (NF-κB) activation is a hallmark of colon tumor growth. Cyclin-dependent kinases (CDKs) are critical cell-cycle regulators, and inhibition of CDK activity has been used successfully as anticancer therapy. Here, we show that the NFE2L3 transcription factor functions as a key regulator in a pathway that links NF-κB signaling to the control of CDK1 activity, thereby driving colon cancer cell proliferation. We found that NFE2L3 expression is regulated by the RELA subunit of NF-κB and that NFE2L3 levels are elevated in patients with colon adenocarcinoma when compared with normal adjacent tissue. Silencing of NFE2L3 significantly decreases colon cancer cell proliferation in vitro and tumor growth in vivo. NFE2L3 knockdown results in increased levels of double homeobox factor 4 (DUX4), which functions as a direct inhibitor of CDK1. The discovered oncogenic pathway governing cell-cycle progression may open up unique avenues for precision cancer therapy.