Chemical synthesis of 2″OMeNAD+ and its deployment as an RNA 2'-phosphotransferase (Tpt1) 'poison' that traps the enzyme on its abortive RNA-2'-PO4-(ADP-2″OMe-ribose) reaction intermediate.

RNA 2'-phosphotransferase Tpt1 catalyzes the removal of an internal RNA 2'-PO4 via a two-step mechanism in which: (i) the 2'-PO4 attacks NAD+ C1″ to form an RNA-2'-phospho-(ADP-ribose) intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Although Tpt1 enzymes are prevalent in bacteria, archaea, and eukarya, Tpt1 is uniquely essential in fungi and plants, where it erases the 2'-PO4 mark installed by tRNA ligases during tRNA splicing. To identify a Tpt1 'poison' that arrests the reaction after step 1, we developed a chemical synthesis of 2″OMeNAD+, an analog that cannot, in principle, support step 2 transesterification. We report that 2″OMeNAD+ is an effective step 1 substrate for Runella slithyformis Tpt1 (RslTpt1) in a reaction that generates the normally undetectable RNA-2'-phospho-(ADP-ribose) intermediate in amounts stoichiometric to Tpt1. EMSA assays demonstrate that RslTpt1 remains trapped in a stable complex with the abortive RNA-2'-phospho-(ADP-2″OMe-ribose) intermediate. Although 2″OMeNAD+ establishes the feasibility of poisoning and trapping a Tpt1 enzyme, its application is limited insofar as Tpt1 enzymes from fungal pathogens are unable to utilize this analog for step 1 catalysis. Analogs with smaller 2″-substitutions may prove advantageous in targeting the fungal enzymes.

STAT3 signaling induced by the IL-6 family of cytokines modulates angiogenesis.

Aberrant angiogenesis is a hallmark of cardiovascular and retinal neovascular disease. The STAT3 signaling pathway represents a potential pharmacological target for these diseases due to its impact on angiogenesis. Surprisingly, some STAT3 activators, such as the IL-6 cytokine family member oncostatin M (OSM), enhance angiogenesis, whereas others, such as ciliary neurotropic factor (CNTF), reduce it. This study aimed to clarify these conflicting effects. In contrast to the anti-angiogenic cytokine CNTF, the pro-angiogenic cytokine OSM was able to activate intracellular signaling pathways beyond the STAT3 pathway, including the ERK and AKT pathways. These differences translated into transcriptomic and metabolic shifts. siRNA-mediated STAT3 knockdown experiments showed a decrease in VEGF-induced endothelial migration and sprouting, enhancing the pro-angiogenic drive of OSM and switching the CNTF response from anti-angiogenic to pro-angiogenic. These effects correlated with a transcriptomic shift representing enhanced STAT1 and ERK activity following STAT3 knockdown, including a compensatory prolonged phosphorylated STAT1 activity. In conclusion, the angiogenic effect of STAT3 appears to be determined by cytokine-induced STAT3 specificity and simultaneous activity of other intracellular signaling pathways, whereas the STAT3 pathway, predominantly recognized for its pro-angiogenic phenotypes, reveals novel anti-angiogenic potential.

CNTF Prevents Development of Outer Retinal Neovascularization Through Upregulation of CxCl10.

Purpose: Ciliary neurotrophic factor (CNTF) is a well-characterized neurotrophic factor currently in clinical trials for the treatment of macular telangiectasia type II. Our previous work showed that CNTF-induced STAT3 signaling is a potent inhibitor of pathologic preretinal neovascular tuft formation in the mouse model of oxygen-induced retinopathy. In this study, we investigated the effect of CNTF on outer retinal and choroidal angiogenesis and the mechanisms that underpin the observed decrease in outer retinal neovascularization following CNTF treatment. Methods: In the Vldlr-/- and laser-CNV mouse models, mice received a one-time injection (on postnatal day [P] 12 in the Vldlr-/- model and 1 day after laser in the Choroidal Neovascularization (CNV) model) of recombinant CNTF or CxCl10, and the extent of neovascular lesions was assessed 6 days posttreatment. STAT3 downstream targets affected by CNTF treatment were identified using quantitative PCR analysis. A proteome array was used to compare media conditioned by CNTF-treated and control-treated primary Müller cells to screen for CNTF-induced changes in secreted angiogenic factors. Results: Intravitreal treatment with recombinant CNTF led to significant reduction in neovascularization in the Vldlr-/- and laser-CNV mouse models. Treatment effect in the Vldlr-/- was long-lasting but time sensitive, requiring intravitreal treatment before P19. Mechanistic workup in vitro as well as in vivo confirmed significant activation of the STAT3-signaling pathway in Müller cells in response to CNTF treatment and upregulation of CxCl10. Intravitreal injections of recombinant CxCl10 significantly reduced outer retinal neovascularization in vivo in both the Vldlr-/- and laser-CNV mouse models. Conclusions: CNTF treatment indirectly affects outer retinal and choroidal neovascularization by inducing CxCl10 secretion from retinal Müller cells.