How Stress Controls the Inflammatory Cytokine Response
Biochemistry
Final Report Abstract
Inflammatory reactions are commonly affected by stress responses. Interleukin-6 signalling is part of the inflammatory response and is stringently regulated by the feedback inhibitor SOCS3 expressed in a short and long isoform. In this project we studied the inhibitory potential of the two SOCS3 isoforms. Furthermore, we analysed the regulation of SOCS3 isoform expression and the role of PKR stress kinase signalling in SOCS3 protein expression. IL-6-induced endogenous expression of both SOCS3 isoforms was found in distinct cell types. Unexpectedly, SOCS3 isoforms show a similar potential for inhibiting IL-6 signalling: Forced expression of either the long or short SOCS3 isoform demonstrated equal inhibitory activity of each isoform but confirmed longer half-life of the short isoform. Our studies of intragenic regulation of SOCS3 isoform expression revealed that (i) the 5’-UTR of SOCS3 mRNA restrains specifically expression of the long SOCS3 isoform, (ii) expression of the long isoform restrains expression of the short isoform, and (iii) in contrast to published data, signalling through the stress kinase PKR does not impact on SOCS3 isoform ratio. Thus, the relative expression of the isoforms depends on intragenic elements yet is independent of PKR signalling. Activation of the RNA-dependent stress kinase PKR leads to phosphorylation of translation initiation factor eIF2α chain. We discovered that interferon-γ and tumor necrosis factor-α genes that encode inflammatory cytokines, as well as the globin genes, use short intragenic RNA elements that activate PKR to control thereby either their splicing or translation in cis. Here, we examined whether SOCS3 mRNA might harbor an intragenic RNA activator of PKR. Our finding is that full-length 2,737-nucleotide mature SOCS3 mRNA potently activates recombinant human PKR. We mapped the SOCS3 RNA activator of PKR to the first exon, nucleotides 1-330 within the 5’-untranslated region of the mRNA. Truncation analysis refined the PKR activator element to nucleotides 141-330. Truncation of a further ten 3’-terminal nucleotides, to position 320, fully abrogated the ability to activate PKR, defining the PKR activator as a 190-nucleotide element. Neither PKR knockdown nor expression of non-phosphorylatable mutant eIF2αS51A affected the splicing efficiency of SOCS3 pre-mRNA. However, eIF2αS51A expression reduced SOCS3 mRNA translation into the more stable short isoform, ΔNSOCS3. Thus, whereas activation of PKR and eIF2α phosphorylation do not regulate SOCS3 intron excision, PKR-mediated eIF2α phosphorylation appears to promote initiation from downstream AUG codon 12, to yield the short, more stable SOCS3 protein isoform. We created a SOCS3 gene in which nucleotides 321-330 are mutated, and are examining its ability to activate PKR and to control SOCS3 protein isoform synthesis.
Publications
- (2015) Interleukin-6: Biology, signaling and strategies of blockade, Cytokine Growth Factor Rev 26, 475-487
Schaper, F., and Rose-John, S.
(See online at https://doi.org/10.1016/j.cytogfr.2015.07.004) - (2016) Interleukin-6 influences stress-signalling by reducing the expression of the mTOR-inhibitor REDD1 in a STAT3-dependent manner, Cellular Signalling 28, 907-916
Pinno, J., Bongartz, H., Klepsch, O., Wundrack, N., Poli, V., Schaper, F., and Dittrich, A.
(See online at https://doi.org/10.1016/j.cellsig.2016.04.004) - (2017) An Ancient Pseudoknot in TNF-α Pre-mRNA Activates PKR, Inducing eIF2α Phosphorylation that Potently Enhances Splicing, Cell Reports 20, 188-200
Namer, L. S., Osman, F., Banai, Y., Masquida, B., Jung, R., and Kaempfer, R.
(See online at https://doi.org/10.1016/j.celrep.2017.06.035) - (2018) Control of mRNA splicing by noncoding intragenic RNA elements that evoke a cellular stress response, The International Journal of Biochemistry & Cell Biology 105, 20-23
Kaempfer, R., Namer, L. S., Osman, F., and Ilan, L.
(See online at https://doi.org/10.1016/j.biocel.2018.09.021)
