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A. Ohlmann, R. Seitz, E. R. Tamm; Mutual Inhibition of Norrin and TGF-ß Signaling in vitro and in vivo. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5967.
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© ARVO (1962-2015); The Authors (2016-present)
To analyze if norrin, a secreted protein that controls capillary formation in the developing retina and acts via the Wnt/ß-catenin pathway, interacts with transforming growth factor (TGF)-ß signaling.
Mink lung epithelial cells (MLEC) and human microvascular endothelial cells (HMEC) were studied. MLEC had been stably transfected with the coding sequences of luciferase under control of the TGF-ß responsive promoter fragment of plasminogen activator inhibitor (PAI) and served as reporter for TGF-ß1 signaling. MLEC and HMEC were incubated with Norrin, Dickkopf (DKK)-1 and/or TGF-ß1 to analyze luciferase activity, mRNA expression of PAI-1, TGF-ß1, TGF-ß-Receptor 1 (TGFß-R1), as well as cell proliferation. In parallel experiments, transgenic mice with an ocular overexpression of Norrin (ßB1-Norrin; Ohlmann et al., J. Neurosci. 2005) or TGF-ß1 (ßB1-TGF-ß1; Flügel-Koch et al., Dev. Dyn. 2002) were mated and double transgenic mice were examined by light and electron microscopy, and real-time RT-PCR.
In TGF-ß1 treated MLEC a substantial increase in the activity of luciferase was observed, an effect that was significantly reduced when TGF-ß1 was added in combination with Norrin. The inhibitory effect of combined Norrin/TGF-ß1 was completely blocked after adding DKK-1, an inhibitor of canonical Wnt/ß-catenin signaling. HMEC that were incubated with TGF-ß1 showed an increase in the expression of PAI mRNA, an effect that substantially decreased upon combined treatment with Norrin/TGF-ß1. After incubation of HMEC with Norrin alone, a pronounced decrease in mRNA for TGF-ß1 and TGFß-R1 was observed. Treatment of HMEC with TGF-ß1 reduced the Norrin-mediated increase in proliferation by about 23%. In the retinae of transgenic ßB1-TGF-ß1 mice the expression of norrin mRNA was significantly decreased when compared to that of wildtype littermates. Vice versa, in retinae of ßB1-Norrin mice the expression of TGF-ß2 mRNA was significantly lower than in wildtype littermates. Finally, the ocular phenotype of ßB1-TGF-ß1 mice such as lack of vitreous body, increase of apoptotic neurons in the retina, and the absence of retinal capillaries was rescued, at least partially, in double transgenic ßB1-TGF-ß1/ßB1-Norrin mice.
Norrin and TGF-ß signaling share a mutual inhibition of their pathways which appears to involve multiple mechanisms including transcriptional inhibition.
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