| Abstract |
Innate immune responses are imperative for the successful defense against endogenous and exogenous threats. However, excess activation can have adverse repercussions and ultimately lead to autoimmune phenomena and irreparable tissue damage. Thus, innate mediators such as monocytes/macrophages have developed a complex system of self regulation that essentially leads to a tolerant and anti-inflammatory phenotype. Among the mechanisms that govern this phenomenon, is the upregulation of molecules that limit signaling downstream of the TLR4 receptor, which recognizes LPS as its ligand.
IRAK-M was first identified in 1999 as a member of the irak family of serine / threonine kinases which enable signal transduction downstream of many TLRs and IL-1β. Originally thought to play a redundant part in this process, it was later shown that in fact is an inactive member, with a non functioning kinase domain. Moreover, ever since 2002 it is considered as one of most important negative regulators of the TLR signaling pathway: primary macrophages from IRAK-M deficient mice, exhibit an intrinsic inability to become tolerant after prolonged LPS stimulation and upon challenging, they are characterized by a profile of increased pro-inflammatory cytokine expression and excess activation of mediators downstream of the TLR signaling complex. In more recent publications, IRAK-M is constantly found upregulated under tolerogenic conditions, in a process that is time and stimulus strength dependent: within boundaries, higher concentrations of TLR ligands and longer incubation periods confer increased IRAK-M mRNA levels. Lastly the role of IRAK-M in regulating innate immunity activation is validated by numerous epidemiological and clinical data.
Despite the relatively good understanding of its mechanism of function, too few are actually known about the actual transcriptional regulation of the IRAK-M gene: many publications exhibit IRAK-M upregulation upon stimulation with a diversity of TLR ligands, however the molecular mechanisms governing this process are still well hidden. Information about transcription factors and signaling pathways that govern its expression is scarce, scattered and fragmented. Roles for PU.1, glucocorticosteroid receptor and SMAD 4 have been suggested but all the evidence is indirect. Also, adiponectin, an adipocyte secreted hormone is able to regulate IRAK-M expression via PI3K/akt and tpl2/ERK dependent mechanisms. Lastly of particular interest are recent publications revealing additional levels in IRAK-M regulation: cellular compartmentalization and trafficking.
The aim of this study is essentially to help in elucidating the mechanisms governing IRAK-M’s expression at the core transcriptional level. We aim to identify key transcription factors that enable this process and decipher their own regulation in the context of inflammation and immune tolerance, events that are timely and functionally discriminated. Lastly it should be noted that this work is a continuation of our efforts in identifying the mechanisms governing PI3K/akt induced tolerance in cells of the innate immunity.
Online data mining revealed that the transcription start site of IRAK-M should be located at a region approximately 90 to 100 base pairs upstream of its translation site (1st exon). This correlates well with cross species homology observations. Moreover, in silico analysis of non coding sequences upstream of the IRAK-M gene revealed that IRAK-M is a TATA-less gene
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and significant clustering of relevant transcription factor binding sites proximally to the putative transcription start region.
We dissected upstream non coding sequences and have found that LPS is able to tranduce IRAK-M expression through an NF-κΒ binding site, located approximately 339 base pairs upstream of the translation start site. NF-κΒ , a key mediator of intracellular LPS signaling, is able to bind directly on this site upon short term LPS stimulation in RAW 264.7 mouse macrophages and transactivate the IRAK-M gene. Moreover we show that IFN-a and IL-10 are unable to upregulate IRAK-M and theorize on putative suppressive effects via direct NF-κΒ-STAT competition on IRAK-M promoter.
Also, we provide further evidence that PI3K/akt signaling affects IRAK-M transcription, as previously stated, by exhibiting interplay with C/EBPβ, an important transcription factor induced upon inflammatory stimuli. LPS is able to promote physical C/EBPβ binding on IRAK-M promoter, in a process largely dependent on PI3K/akt signaling, as treatment with wortmanin –a PI3K inhibitor is able to abrogate this phenomenon.
Thus, these data point towards an old and basic theme in immunobiology: Signaling pathways essentially lead their own inactivation. Here we show that TLR4 signaling promotes its inactivation, by utilizing two of the transcription factors that confer its inflammatory effect.
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