Date Published: March 21, 2017
Publisher: Public Library of Science
Author(s): Samira Ghorbani, Farideh Talebi, Sedigheh Ghasemi, Ali Jahanbazi Jahan Abad, Mohammed Vojgani, Farshid Noorbakhsh, Muzamil Ahmad.
MicroRNAs are small noncoding RNAs, which regulate the expression of protein coding transcripts through mRNA degradation or translational inhibition. Numerous reports have highlighted the role of miRNAs in regulating cell death pathways including the expression of genes involved in the induction of apoptosis. Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine which can send pro-death signals through its receptor TNFR1. Diverse adaptor molecules including DENN/MADD adaptor protein have been shown to modulate TNF-α pro-death signaling via recruitment of MAP kinases to TNFR1 and activation of pro-survival NFκB signaling. Herein, we investigated the role of microRNA-181 (miR-181) in regulating DENN/MADD expression levels and its subsequent effects on TNF-α-induced cell death. Using bioinformatics analyses followed by luciferase reporter assays we showed that miR-181 interacts with the 3’ UTR of DENN/MADD transcripts. miR-181 overexpression also led to decreased endogenous DENN/MADD mRNA levels in L929 murine fibroblasts. Flow cytometric analysis of miR-181 transfected cells showed this miRNA accentuates mitochondrial membrane potential loss caused by TNF-α. These findings were associated with enhanced apoptosis of L929 cells following TNF-α treatment. Overall, these data point to the potential role of miR-181 in regulating TNF-α pro-death signaling, which could be of importance from pathogenesis and therapeutic perspectives in inflammatory disorders associated with tissue degeneration and cell death.
TNF-α is a proinflammatory cytokine, which plays critical roles in diverse inflammatory disorders [1, 2]. In addition to regulating inflammation, TNF-signaling might affect cell viability through pro-death and/or pro-survival signaling. TNFR1, a ubiquitously expressed TNF receptor, has been demonstrated to be involved in both pro-death and pro-survival TNF signaling in different cell types [3, 4]. Different studies have shown the involvement of various adaptor molecules in determining the nature of signals sent into the cells following TNF binding to TNFR1 [5, 6]. It is believed that the binding of TNFR-associated death domain (TRADD) molecule to TNFR1 leads to the recruitment of Fas-associated death domain (FADD) protein which promotes apoptosis, while the interaction with receptor-interacting protein (RIP) and TNFR-associated factor (TRAF2) can lead to cell survival. Indeed, binding of TRAF2 is believed to trigger the activation of pro-survival MAP kinases as well as the NFκB signaling [7, 8]. While TNF is widely known for its ability to induce cell death in the context of inflammation, this alternative signaling can explain the protective effects of TNF in some studies where TNF knock out or blockade has led to increased cell/tissue injury . Search for mechanisms which can switch the TNF signaling from pro-apoptotic to pro-survival has led to the identification of a few adaptor molecules, including DENN/MADD (differentially expressed in normal versus neoplastic/ MAPK activating death domain). DENN/MADD is a death domain (DD)-containing protein which has been illustrated to interact with TNFR1, competing with the binding of TRADD and skewing the signaling pathway towards cell survival .
TNF-α in known to promote cell death and apoptosis in a variety of inflammatory conditions including cancer and neurodegenerative diseases, however, its cytoprotective effects has also been shown in other studies [9, 27–30]. While different TNF receptors might send different signals into the cells, part of the diverse effects of TNF seem to be dependent on the recruitment of the different adaptor molecules to one ubiquitously expressed receptor, i.e. TNFR1, which can switch the signaling between pro-death and pro-survival pathways [7, 8, 10]. The adaptor molecule DENN/MADD exerts an anti-apoptotic role by binding to the TNFR1 death domain. DENN/MADD can directly activate pro-survival MAP kinases or enhance the effect of TRAF2 signaling, a competitor of TRADD [10, 31, 32]. Studies regarding the role of DENN/MADD in human disease have mostly confirmed its pro-survival properties within the context of cancer. An early study by Lim et al, showed that DENN/MADD silencing by siRNAs induce apoptosis in several mammalian cancer cell lines . Later studies reported the involvement of DENN/MADD in promoting cancer cell survival in the context of leukemia , neuroblastoma, thyroid and lung cancer [35–38]. DENN/MADDs potential role in the pathogenesis of neurodegenerative diseases has also been investigated in recent years [39, 40]. A research on neuronal cell death in Alzheimer’s disease has shown significant down-regulation of DENN/MADD and enhanced hippocampal neuronal death following DENN/MADD silencing . Reduced endogenous DENN/MADD expression in brain is believed to perturb neuronal survival in AD patients and transgenic mouse models of disease [39, 40]. We have also observed decreased DENN/MADD expression levels in the nervous system of EAE mice, an experimental animal model of multiple sclerosis (S3 Fig). These reports point to a potential cytoprotective effect for DENN/MADD through inhibiting TNF-induced apoptotic cell death. In the current study, we show experimental evidence pointing to the regulation of DENN/MADD by miRNA-181 through luciferase assays and transfection experiments. Among the microRNAs which are predicted to target DENN/MADD, we focused on miR-181 family members because of their implication in regulating apoptosis pathways and their broad conservation across species. Studies on human glioma and glioma cell lines have shown decreased expression of miR-181 family members together with apoptosis induction and tumor growth inhibition following miRNA overexpression [41, 42]. Other studies on astrocytes have shown increased resistance to apoptosis following miR-181 reduction likely through altered expression of Bcl-2 family members. . Aberrant expression of miR-181 in neurodegenerative disease like multiple sclerosis has also been reported in previous studies [15, 17]. Gene ontology analysis on the putative targets of miR-181 also indicates the involvement of this microRNA in cellular death processes (S4 Fig).