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Cavit Agca, Ghislaine Traber, Christian Caprara, Christel Beck, Isabelle Marie-Paule Meneau, Christian Grimm; Cis-regulation of Lif mRNA stability in Muller cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5588.
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Leukemia inhibitory factor (LIF) is an important endogenous factor for photoreceptor protection. Lif is known to be upregulated both in induced and inherited disease models of photoreceptor degeneration only in a subset of Muller cells, however little is known about its regulation during a photoreceptor injury. Since Lif is induced in Muller cells in response to photoreceptor injury, we studied its regulation in Muller cells both in vivo and in vitro and elucidated cis-regulatory elements that are responsible for the post-transcriptional regulation of Lif transcripts as well as the upstream factors that are potentially involved. We have also generated a BAC-transgenic mouse expressing a LIF::EGFP fusion protein to study the Lif-expressing subset of Muller cells in vivo.
Reporter constructs, which carry fragments of the Lif 3'UTR fused to the luciferase gene are utilized to test the effects on mRNA stability. The rMC-1 cells were transfected with unstable Luciferase expressing constructs. Relative Luciferase levels were determined by Luciferase assays. Mice were injected intravitreally with recombinant TNF-alpha and an inhibitor of P38 MAPK to determine their effects on Lif expression in vivo. Retinas were collected and Lif levels were determined by RT-PCR.
Mouse Lif has several AU-rich elements that are dispersed within its 3` UTR. Two of the tested elements induced a significantly higher instability, which was verified by deletion and insertion experiments. Sequence comparison of these non-coding regions showed a strong conservation between vertebrates, suggesting a common mechanism for Lif regulation. These results are also consistent with our previous (unpublished) data showing that the Lif mRNA turnover is influenced by H2O2 in rMC-1 cells. Moreover, intravitreal injections of TNF-alpha induced Lif upregulation in a similar fashion to our in vitro studies and blocking the P38 MAPK pathway inhibits this upregulation.
These results strongly argue that mouse Lif mRNA is at least partially regulated by its 3`UTR region and that P38 MAPK pathway may be involved through a redox-dependent pathway. The identified AU-rich elements which are responsible for the high instability of Lif transcripts reside in highly conserved regions among vertebrates. This suggests that the post-transcriptional regulation of Lif transcripts is evolutionarily conserved and that these elements may also be functional in other vertebrates including humans. Since Lif has been shown to be an integral part of retinal endogenous protective pathway , these results will contribute to a better understanding of neuroprotection in retinal diseases.
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