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NA WU, Joseph Wiseman, Yuan Lei, Karen Eastlake, Xinghuai Sun, G Astrid Limb; Modulation of the Notch and Wnt signalling by TGF- β in adult human Müller stem cells.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1373.
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© ARVO (1962-2015); The Authors (2016-present)
The transforming growth factor β (TGF- β) and the Wnt, Notch signalling pathways all play crucial roles in many biological processes, including embryonic development, cell fate differentiation and cell proliferation. There is evidence for a crosstalk between the Notch and Wnt pathways during retinal development, but it is not known whether co-activation of these pathways may occur in adult human Müller stem cells (hMSCs) or whether these can be modulated by TGF- β.It was therefore the aim of this study to investigate the effect of this cytokine on the expression of molecules involved in these two pathways
Human Müller stem cells were cultured on matrigel using DMEM containing foetal calf serum (FCS). hMSCs were cultured in the absence or presence of the γ-secretase inhibitor DAPT plus bFGF to induce their differentiation into RGCs. Exogenous TGF- β1 or TGF- β3 were added to the differentiating or control cells which were cultured for up to 7 days. mRNA and protein extracted from these cells were examined by RT-PCR, western-blot and immuno-staining techniques for the expression of molecules of the Notch and Wnt signalling pathways. The hexosaminidase assay and Ki67 immuno-staining were used to test the effect of TGF- β on cell proliferation.
Notch inhibition by DAPT caused down-regulation in gene expression of Hes1 and Wnt 2b. Similarly, TGF- β1 alone caused a decrease in gene and protein expression of the Notch downstream target Hes1 and the Wnt signalling ligand Wnt2b, while inducing an increase of the canonical Wnt signalling intracellular component β-catenin. Down-regulation of these genes by DAPT was not modified by addition of TGF- β1 or TGF- β3 to the cultured cells. It was also observed that both TGF- β1 and TGF- β3 had a similar effect on the inhibition of cell growth.
These results provide evidence that TGF- β directly induces down-regulation of the Notch target Hes1 and the Wnt signalling ligand Wnt2b, as well as upregulation of β-catenin. Furthermore, down-regulation of Notch by DAPT results in down-regulation of Wnt2b, suggesting a crosstalk between these two signalling pathways. However, TGF- β does not have additive effects on the down-regulation of Hes1 and Wnt2b by DAPT. The observations suggest that these two molecules may activate similar intracellular pathways mediating neural differentiation of Müller stem cells.
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