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Delphine M. Bonnet Wersinger, Sarah E. Collins, Jun Wan, Cynthia A. Berlinicke, Donald J. Zack; Exploration of the mechanism by which Spleen Tyrosine Kinase (SYK) inhibitor R406 promotes rhodopsin expression. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1319.
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© ARVO (1962-2015); The Authors (2016-present)
Through a small molecule screen we identified the SYK inhibitor R406 as being able to stimulate rhodopsin reporter activity in cultured murine primary retinal cells. Since our data suggested that R406’s effect on rod differentiation was independent of its SYK inhibitory activity, we sought to determine the signaling pathway(s) mediating its photoreceptor activity. Based on other known R406 targets, we concentrated on the Jak and Flt3 pathways.
Dissociated retinal cells were prepared from postnatal C57BL/6J, RhoGFP knock-in and Flt3-/- mice. Cultures were treated with R406 (Rigel Pharmaceuticals, San Francisco), JAK or FLT3 pharmacological inhibitors (EMD Chemicals). Rod photoreceptor differentiation was quantified through rhodopsin reporter assays with Rho-EGFP knock-in or secreted luciferase reporters. Total RNA and proteins were prepared from R406 treated retinal cells and assayed by QPCR and Western-Blot analysis, respectively. R406 induced transcriptome changes obtained by exon arrays were confirmed by QPCR.
Administration of increasing doses of FLT3 inhibitors did not stimulate rhodopsin GFP or luciferase reporter expression. In addition, retinal cultures from Flt3-/- mice did not show enhanced rhodopsin reporter expression in luciferase assays. Interestingly, Jak mediated phosphorylation of STAT3 was dose-dependently reduced by R406. In addition, R406 pre-treatment prevented CNTF dose-dependent activation of STAT3. Moreover, the JAK inhibitor I competed with R406 in terms of its ability to induce rhodopsin reporter expression. Muller glial specific RNA transcripts were identified as 31% of the genes regulated by R406 in the microarray study. Quantitative PCR verified significant decrease of several glial genes, including Cryab, GFAP, Msn, Rgs5 and Vim. The major Notch target genes Hes1 and Hes5 were significantly downregulated in postnatal retinal cells after R406 treatment.
The effect of R406 on rhodopsin expression in primary retinal cultures does not appear to be mediated by inhibition of FLT3 or SYK activity. Our data suggests that Notch inhibition and modulation of Jak/STAT3 signaling may be involved in R406’s activity, and that there may be a relationship with glial cell differentiation. Further definition of these pathways may provide novel insights into photoreceptor development.
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