ABSTRACT
IL-17 is a proinflammatory cytokine that promotes the expression of different cytokines and chemokines via the induction of gene transcription and the posttranscriptional stabilization of mRNAs. In this study, we show that IL-17 increases the half-life of the Zc3h12a mRNA via interaction of the adaptor protein CIKS with the DEAD box protein DDX3X. IL-17 stimulation promotes the formation of a complex between CIKS and DDX3X, and this interaction requires the helicase domain of DDX3X but not its ATPase activity. DDX3X knockdown decreases the IL-17-induced stability of Zc3h12a without affecting the stability of other mRNAs. IKKε, TNFR-associated factor 2, and TNFR-associated factor 5 were also required to mediate the IL-17-induced Zc3h12a stabilization. DDX3X directly binds the Zc3h12a mRNA after IL-17 stimulation. Collectively, our findings define a novel, IL-17-dependent mechanism regulating the stabilization of a selected mRNA.
Subject(s)
DEAD-box RNA Helicases/metabolism , Gene Expression Regulation , Interleukin-17/metabolism , RNA Stability , Ribonucleases/genetics , Transcription Factors/genetics , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/metabolism , Adaptor Proteins, Signal Transducing , Gene Expression Regulation/drug effects , Humans , I-kappa B Kinase/metabolism , Interleukin-17/pharmacology , Multiprotein Complexes/metabolism , Protein Binding/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , TNF Receptor-Associated Factor 2/metabolism , TNF Receptor-Associated Factor 5/metabolismABSTRACT
CONTEXT: We have previously identified neutrophil gelatinase-associated lipocalin (NGAL) as one of the genes mediating the oncogenic activity of nuclear factor-κB in human anaplastic thyroid carcinomas (ATCs). OBJECTIVES: To further investigate the role of NGAL in thyroid cancer, we established NGAL knocked-down and NGAL overexpressing ATC cell lines. RESULTS: We found that the ability of NGAL knocked-down cells to degrade Matrigel in a transwell invasion assay and to form lung metastasis in nude mice was decreased. Because NGAL binds matrix metalloproteinase-9 (MMP-9), to form a macromolecular complex involved in the regulation of metastatic spread of cancer cells and given the strong expression of both genes in tissue specimens from human ATCs, we analyzed the MMP-9 enzymatic activity in NGAL-null ATC cells. Enzymatic immunoassays show that MMP-9 activity is reduced in NGAL-null ATC cells, even if its expression is not affected by NGAL inhibition. Ectopic expression of NGAL in an ATC cell line not expressing NGAL determines an increase of its metastatic property. The use of a mutated form of NGAL, unable to bind MMP-9, has no positive effect on the invasive potential of ATC cells and does not improve the MMP-9 enzymatic activity. CONCLUSIONS: Our results indicate NGAL as a novel target of nuclear factor-κB prometastatic activity in thyroid cancer through enhancement of MMP-9 enzymatic activity.
Subject(s)
Acute-Phase Proteins/physiology , Lipocalins/physiology , Proto-Oncogene Proteins/physiology , Thyroid Neoplasms/genetics , Thyroid Neoplasms/pathology , Acute-Phase Proteins/antagonists & inhibitors , Acute-Phase Proteins/genetics , Acute-Phase Proteins/metabolism , Animals , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , HEK293 Cells , Humans , Lipocalin-2 , Lipocalins/antagonists & inhibitors , Lipocalins/genetics , Lipocalins/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Nude , NF-kappa B/metabolism , NF-kappa B/physiology , Neoplasm Invasiveness , Neoplasm Metastasis , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA, Small Interfering/pharmacology , Thyroid Carcinoma, Anaplastic , Tumor Cells, CulturedABSTRACT
The demographic tendency in industrial countries to delay childbearing, coupled with the maternal age effect in common chromosomal aneuploidies and the risk to the fetus of invasive prenatal diagnosis, are potent drivers for the development of strategies for noninvasive prenatal diagnosis. One breakthrough has been the discovery of differentially methylated cell-free fetal DNA in the maternal circulation. We describe novel bisulfite conversion- and methylation-sensitive enzyme digestion DNA methylation-related approaches that we used to diagnose Turner syndrome from first trimester samples. We used an X-linked marker, EF3, and an autosomal marker, RASSF1A, to discriminate between placental and maternal blood cell DNA using real-time methylation-specific PCR after bisulfite conversion and real-time PCR after methylation-sensitive restriction digestion. By normalizing EF3 amplifications versus RASSF1A outputs, we were able to calculate sex chromosome/autosome ratios in chorionic villus samples, thus permitting us to correctly diagnose Turner syndrome. The identification of this new marker coupled with the strategy outlined here may be instrumental in the development of an efficient, noninvasive method of diagnosis of sex chromosome aneuploidies in plasma samples.