Expression of Wnt signaling pathway genes during erythroid terminal differentiation (ETD)
MetadataShow full item record
Background: Erythropoiesis is a complex multistep process whereby nucleated erythroid progenitor cells differentiate into enucleated red blood cells. The processes of proliferation, differentiation and apoptosis are balanced in erythroid progenitors to produce mature erythrocytes. Wnt signaling pathways are conserved throughout evolution and influence cell proliferation, migration, polarity and differentiation. This study examined whether the Wnt signaling pathway is involved in erythroid terminal differentiation (ETD). Methods: Murine splenic erythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells) were induced to differentiate by treatment with erythropoietin (EPO). Quantitative real time polymerase chain reaction focused arrays containing eighty four genes involved in Wnt signaling pathways were used to compare gene expression at before and after 48h of EPO induced differentiation. SDS-PAGE and western blotting analyses were performed on the five most highly up-regulated Wnt related genes to verify the results obtained by RT-PCR array. Results: RT-PCR arrays identified statistically significant upregulation of 13 Wnt signaling pathway-related genes during ETD. The expression of Wnt4 was up-regulated 25 fold. Western blot analysis of Wnt4 protein showed a single sharp intense band calculated to be 37.3 kDa. ICAT and c-Jun were found to be upregulated 9 fold each. Western blot analysis of ICAT showed two bands of molecular weight of 160.2 kDa and 67.7 kDa for only treated FVA cells. Western blot analysis of c-Jun protein did not yield any bands in either control or treated FVA cell samples. mRNA expression of Ep300 was up-regulated 8 fold while Daam1 was up-regulated about 6 fold in differentiating FVA cells. Western blot analysis of Ep300 protein showed a single band of molecular weight 97 kDa in control FVA cell samples while 48 hour FVA sample showed the presence of three very light bands with molecular weights corresponding to 172.2 kDa, 133.4 kDa and 97 kDa. While in case of Daam1, western blot analysis of the control FVA cell samples showed two bands of 97 kDa and 81 kDa while 48 hour sample showed only one band at around 124 kDa. Other upregulated genes included: Fzd1, Csnk1d, Ctnnb1, Fzd5, Wnt2, Fbxw2, Csnk1a1 and Nkd1. Discussion: Wnt4 participates in regulating the cellular fate hence we believe that Wnt4 may be involved in actin polymerization during ETD via PCP-like non-canonical Wnt signaling pathway. ICAT has been shown to act as a negative regulator of Wnt signaling pathway and plays an important role in monocytic differentiation. Increased mRNA expression in differentiating FVA cells suggest that ICAT may prevent the canonical Wnt signaling by binding to β-catenin. c-Jun plays a role in cell proliferation and acts as positive and negative regulator of apoptosis. During ETD, c-Jun may be delaying apoptosis of differentiating FVA cells and promoting proliferation of cells. Ep300 plays a role in transcription regulation. Ep300 may be carrying out chromatin remodeling which is essential for ETD. Daam1 plays a critical role in mediating non-canonical Wnt signaling pathway and actin polymerization. Significance: Results of this study suggest that genes involved in the Wnt signaling pathway may play a role in the events that occur during ETD. Wnt signaling has previously been shown to be important in the expansion of hematopoietic stem cells. This study, to the best of our knowledge, is the first evidence that Wnt signaling may be involved in the latest stages of ETD in differentiating erythroid cells other than the self-renewal.