Abstract
We reported that gene-selective formation of facultative heterochromatin silences transcription of acute inflammatory genes during endotoxin (LPS) tolerance, according to function. We discovered that reversal of the epigenetically silenced transcription restored mRNA levels but not protein synthesis. Here, we find that translation repression of tumor necrosis factor-alpha (TNFalpha) occurs independent of transcription silencing during LPS tolerance. The process required to disrupt protein synthesis followed Toll-like receptor 4 (TLR4)-dependent induction of microRNA (miR)-221, miR-579, and miR-125b, which coupled with RNA-binding proteins TTP, AUF1, and TIAR at the 3'-untranslated region to arrest protein synthesis. TTP and AUF1 proteins linked to miR-221, whereas TIAR coupled with miR-579 and miR-125b. Functional inhibition of miR-221 prevented TNFalpha mRNA degradation, and blocking miR-579 and miR-125b precluded translation arrest. The functional specificity of the TNFalpha 3'-untranslated region was demonstrated using luciferase reporter with mutations in the three putative miRNA binding sites. Post-transcriptional silencing was gene-specific, because it did not affect production of the IkappaBalpha anti-inflammatory protein. These results suggest that TLR4-dependent reprogramming of inflammatory genes is regulated at two separate and distinct levels. The first level of control is mediated by epigenetic modifications at the promoters that control transcription. The second and previously unrecognized level of control is mediated by TLR4-dependent differential expression of miRNAs that exert post-transcriptional controls. The concept of distinct regulation of transcription and translation was confirmed in murine sepsis. We conclude that transcription- and translation-repressive events combine to tightly regulate pro-inflammatory genes during LPS tolerance, a common feature of severe systemic inflammation.
Highlights
Inflammation development and progression are influenced by genetic, epigenetic, and environmental factors [1]
Mature miRNAs are loaded onto a ribonucleoprotein complex, known as miRNA-induced silencing complex, where they act as guiding molecules to deliver the complex to target mRNA via binding to complementary sequences in the 3ЈUTR, resulting in mRNA degradation and/or translational repression [15]. miRNAs usually base pair to target mRNA with imperfect complementarity, resulting in translational inhibition, whereas perfect base-pairing induces target mRNA degradation [15, 16]
Reversing transcription silencing by RelB knockdown restored TNF␣ mRNA to near normal levels seen in responsive (R) cells, we did not detect protein even after 4 – 6 h of re-stimulation
Summary
The inciting and evolving features of SSI conform to the network motif concept of type 1 incoherent feed-forward loops [11], in which a transcription activator X (e.g. NF-B p65) controls a target gene Z (e.g. TNF␣) and sequentially activates a transcription repressor Y (e.g. RelB) of the target gene, producing physiologic adaptation, for example tolerance. Mature miRNAs are loaded onto a ribonucleoprotein complex, known as miRNA-induced silencing complex (miRISC), where they act as guiding molecules to deliver the complex to target mRNA via binding to complementary sequences in the 3ЈUTR, resulting in mRNA degradation and/or translational repression [15]. We performed a computational miRNA target prediction algorithm and identified twenty miRNAs with sequences complementary to the TNF␣ 3ЈUTR, as a model of post-transcriptional repression of pro-inflammatory genes in LPS tolerance. Our studies provide the first evidence linking differential changes in TLR-induced miRNA expression to LPS tolerance, which serves as a cell model of SSI gene reprogramming [2, 19], and suggest that both disruption of transcription and translation independently assure gene-specific silencing during the endotoxin tolerance/evolving phase of SSI
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