| Abstract |
We have previously shown that activation of CD40 induces IRF1 (1), a member of the IRF transcription family which acts as a master regulator of genes involved in innate and adaptive immune responses (2). In this study we explore the mechanisms of two separate pathways leading to IFN-β or TPL2 induction downstream of the CD40/NF-κB/IRF1 axis. IFN-β and the CD40L (CD154) share important roles in the antiviral and antitumor immune responses which led us to the hypothesis that IFN-β could be a physiological target of CD40 signaling. Data presented in the first section of this study demonstrate that CD40 receptor occupancy results in IFN-β upregulation in tumor cells via a novel mechanism that requires integration of canonical and non canonical NF-κB signaling pathways. We show that the binding of RelA (p65) and p52 NF-κB subunits to the IFN-β promoter directly contributes to gene expression and that CD40-mediated IFN-β transactivation ceases only upon impairment of both pathways. These findings suggest that whereas canonical and non-canonical NF-κB pathways operate independently, they converge to coregulate IFN-β gene expression. In line with this observation, overexpression of p52 alone is capable of inducing IFN-β promoter activity, and blockade of the canonical NF-κB pathway does not interfere with p52 binding to the IFN-β promoter. Importantly, however, the rapid, RelA-dependent de novo synthesis of IRF1 and Viperin also allows for the recruitment of IRFs, providing a tremendous enhancement of IFN-β gene induction. Overall we describe an unconventional “feed-forward” mechanism of signal transduction, leading to IFN-β upregulation which is orchestrated by the parallel engagement of canonical and non-canonical NF-κB pathways. These data define a novel link between CD40 signaling and IFN-β expression and provide a telling example of how signal propagation can be exploited to ensure efficient regulation of gene expression.
Tumor Progression Locus 2 (TPL2), a kinase implicated in the regulation of innate and adaptive immune responses, is known to mediate ERK induction downstream of CD40. RNA microarray experiments showed that both TPL2 and IRF1 are upregulated
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downstream of CD40, IL-6 and TCR receptors. Also ChIP sequencing experiments revealed direct binding of IRF1 upstream of TPL2 gene locus. These observations lead us to the hypothesis that CD40 induces TPL2 expression via IRF1. Data presented in the second section of this thesis, show that IRF1 orchestrates TPL2 induction downstream of CD40 and IFN-γ leading to delayed ERK phosphorylation in cancer cells. TPL2 mRNA and protein levels were significantly increased by CD40 ligation and IFN-γ treatment, whereas siRNA-mediated knockdown of IRF1 eliminated TPL2 induction downstream of both pathways. Moreover, overexpression of wild type IRF1 was sufficient to induce increased protein levels of TPL2 in contrast with a dominant negative IRF1 mutant. ChIP assays showed in vivo binding of IRF1 at sequences flanking the 2 IRF-E motifs found on TPL2 promoter region. Reporter assays of two different regions of TPL2 promoter containing one or both IRF-E motifs verified the functional role of IRF-1 mediated TPL2 transcriptional activation. Interestingly, enhanced ERK phosphorylation observed following CD40L and IFN-γ treatment was disrupted using a small chemical kinase inhibitor and siRNA targeting TPL2. These data suggest a novel mechanism of TPL2 induction depending on its transcriptional activation by IRF1. Newly synthesized TPL2 kinase mediates a delayed ERK phosphorylation which acts as a second wave of signal transduction downstream of both CD40 and IFN-γ. Both signals play an important role in innate immune responses, thus it is possible that this mechanism can act as a regulator of their intensity and duration. More experiments are necessary to clarify the physiological role of this novel pathway. Interestingly recent studies revealed that TPL2 acts as a tumour suppressor and its expression is reduced in human lung cancer samples compared to surrounding normal tissue (3,4), while CD40 is known to induce apoptosis and targeted immune responses to cancer cells, suggesting a possible functional link of TPL2 induction in cancer cells.
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Μελέτη μηχανισμών μεταγραφικής ρύθμισης από τον υποδοχέα CD40
