Análisis de una arritmia

The Notch and Wnt signalling pathways have been shown to be involved in Müller glia proliferation and differentiation across various species, including zebrafish and small mammals (Wan et al., 2012, Ramachandran et al., 2011, Del Debbio et al., 2010). In addition, it has been demonstrated that activation of the Notch intracellular domain can inhibit the canonical Wnt signalling pathway possibly through expression of HES1 (Deregowski et al., 2006). Thus, we investigated the effect of Notch inhibition on the canonical and non-canonical Wnt signalling components in various hMSC lines.

Culture of MIO-M1 cells with 0.5 μM RO4929097 resulted in a significant decrease in WNT2B mRNA expression (p<0.01) as compared to cells cultured with medium alone (Fig 2.9A). Additionally, western blot analysis of whole cell lysates of MIO-M1 cells cultured with RO4929097 demonstrated a decrease in WNT2B protein expression (p<0.05) upon Notch inhibition as compared to control cells (Fig 2.9B).

When three different cell lines (6426, 6387 and 6391) were cultured with the Notch inhibitor RO4929097, a significant decrease in WNT2B mRNA expression was observed in the cell line 6387 (p<0.05) when compared to the control cells (Fig 2.9).

However, RO4929097 did not cause any changes in the mRNA expression of this gene in the other cell lines 6426 and 6391 as compared to control cells (Fig 2.9).

Corresponding to the decrease observed in WNT2B mRNA and protein expression by 0.5 μM RO4929097 in MIO-M1 (Fig 2.9) and mRNA expression in 6387 hMSC cell line (Fig 2.10) respectively, Notch inhibition also caused a significant decrease

in the mRNA expression of the canonical Wnt signalling targets WISP-1 (p<0.01) and AXIN2 (p<0.05) in MIO-M1 cells, whilst DKK1 mRNA expression was increased in this hMSC line as compared to control cells (2.11A). In 6387 cell line, a significant decrease in DKK1 (p<0.05) and WISP-1 (p<0.05) mRNA expressions were caused by RO4929097 when compared to control cells (Fig 2.11B). However, no significant change was observed in mRNA coding for AXIN2 by RO4929097 in this cell line (Fig 2.11B). When three different hMSC lines MIO-M1, 6426 and 6387 were cultured with 0.5 μM RO4929097 for the examination of non-canonical Wnt signalling ligand WNT5B, there was a significant decrease in WNT5B mRNA expression (p<0.001) in MIO-M1 cells as compared to the control cells (Fig 2.12). However, 0.5 μM RO4929097 did not modify the WNT5B mRNA expression in hMSC lines 6426 and 6387 as compared to controls (Fig 2.12).

The results suggest that Notch inhibition by RO4929097 inhibits the components of the canonical Wnt signalling including WNT2B, AXIN2 and WISP-1 in hMSC.

Additionally, non-canonical Wnt signalling ligand WNT5B is also downregulated by inhibition of the Notch signalling in hMSC. However, similar to the variation seen in different hMSC lines in the downregulation of HES1 by this γ-secretase inhibitor, differences in the downregulation of the WNT components were also observed in the different examined hMSC lines.

Figure 2.9 Effect of Notch inhibition on the canonical Wnt signalling ligand WNT2B in MIO-M1 cells. (A) Notch inhibition in MIO-M1 cells by RO4929097 induced downregulation of WNT2B mRNA expression in these cells; n=3. Student’s t-test; **p<0.01 v. control. (B) Western blot analysis of cell lysates from MIO-M1 cells showed a mark decrease of WNT2B intracellular protein in cells treated with RO4929097 at a concentration of 0.5 μM as compared to control cells; n=3. Student’s t-test, *p<0.05 v. control. . Histograms represent the mean + SEM of the optical density of gel bands normalized to β-actin. Representative gel bands are shown above histograms.

Figure 2.5 Effect of Notch inhibition on the canonical Wnt signalling ligand WNT2B in MIO-M1 cells. (A) Notch inhibition in MIO-M1 cells by RO4929097 induced downregulation of WNT2B mRNA expression in these cells; n=3.

Student’s t-test; **p<0.01 v. control. (B) Western blot analysis of cell lysates from MIO-M1 cells showed a mark decrease of WNT2B intracellular protein in cells treated with RO4929097 at a concentration of 0.5 μM as compared to control cells; n=3. Student’s t-test, *p<0.05 v. control. . Histograms represent the mean + SEM of the optical density of gel bands normalized to β-actin. Representative gel bands are shown above histograms.

WNT2B β-actin

Control RO4929097

Figure 2.10 Effect of Notch inhibition on the canonical Wnt signalling ligand WNT2B in various Müller stem cell lines (6426, 6387 and 6391). Notch inhibition by 0.5 μM RO4929097 induced downregulation in the expression of WNT2B mRNA in the 6387 cell- line, whilst there were no changes were observed in the cell lines 6426 and 639; n=3-4.

Student’s t-test; *p<0.05 v. control. Histograms represent the mean + SEM of the optical density of gel bands normalized to β-actin. Representative bands are shown above histograms.

Figure 2.11 Effect of Notch inhibition on the canonical Wnt signalling component β-catenin and Wnt target genes DKK1, WISP-1 and AXIN2 in in Müller stem cell lines (MIO-M1, 6387 and 6391). A) In MIO-M1 cell line, Notch inhibition by 0.5 μM RO4929097 caused upregulation in mRNA expression of DKK1. In contrast, RO4929097 used at the same concentration caused downregulation in mRNA expression of WISP-1 and AXIN2 in these cells; n=3-4. Student’s t-test; *p<0.05 v. control. B) Culture of 6387 cells with RO4929097 at the same concentration caused downregulation in mRNA expressions of DKK1 and WISP-1 in these cells but did not alter AXIN2 mRNA expression; n=3-4. Student’s t-test; *p<0.05 v. control.

Figure 2.12 Effect of Notch inhibition on the non-canonical Wnt signalling ligand WNT5B in the Müller glia stem cell lines MIO-M1, 6426 and 6387. Notch inhibition by 0.5 μM RO4929097 caused a decrease in mRNA expression of WNT5B in the MIO-M1 cell line, whilst it did not induce changes in the 6426 and 6387 cells; n=3. Student’s t-test; *p<0.05 v.

control. Histograms represent the mean + SEM of the optical density of gel bands normalized to β-actin. Representative bands are shown above histograms.