In order to understand how hematopoiesis

In order to understand how hematopoiesis is regulated, it is necessary not only to understand the different signals emanating from the niche (Anthony and Link, 2014), but also to comprehend the integration of these signals by HSCs. Canonical Wnt signaling has been related to the regulation of HSCs homeostasis (Reya et al., 2003), and it has been reported that a switch toward a non-canonical Wnt signaling causes stem-cell aging (Florian et al., 2013). β-catenin is the nuclear effector of canonical Wnt signaling, and it also behaves as a cell adhesion molecule owing to its interaction with cadherins (Valenta et al., 2012). Although it has been shown that Wnt/β-catenin is required for hematopoiesis in Xenopus (Tran et al., 2010), the role of β-catenin in mammalian hematopoiesis remains highly controversial (Luis et al., 2012). We have recently shown that the protein tyrosine phosphatase PTPN13 regulates β-catenin stability and function during in vitro megakaryopoiesis (Sardina et al., 2014). Our results also show that PTN13 is stabilized upon Wnt signaling activation, suggesting that PTPN13 is another important player in the context of canonical Wnt signaling (Sardina et al., 2014). The deficiency of PTPN13 in mice increases the in vitro differentiation of CD4+ T cells toward Th1 and Th2 (Nakahira et al., 2007), which together with our results (Sardina et al., 2014) suggests that PTPN13 may be an important regulator during hematopoiesis.
Results
Discussion Understanding the mechanisms that govern the balance between self-renewal and differentiation or between cell-cycle progression and quiescence in HSCs is essential. These processes are regulated by the different signals received by HSCs from the BM niche. Accordingly, it is crucial to understand how these inputs are processed by HSC intracellular signaling. In this work, we show that the levels of PTPN13 and β-catenin are important for regulating HSCs quiescence and cell adhesiveness, and our results suggest that the regulation of the levels of these two proteins by different extracellular signaling pathways might be essential for the regulation of HSC biology. Moreover, in agreement with previous reports (Sardina et al., 2014; Nakahira et al., 2007; Mulroy et al., 2003; Xu et al., 2003), our results also suggest the involvement of these two proteins in megakaryocytic and lymphoid maturation. In future studies, we shall analyze this issue in greater depth. The in vivo silencing of PTPN13 or β-catenin reduced the total number of engrafted GFP+ UNC2025 in BM. However, it led to an increased frequency of both GFP+ LT-HSCs and GFP+ ST-HSCs. HSCs have a low proliferation rate (Pietras et al., 2011), and hence the enrichment in HSCs would explain the reduced number of engrafted GFP+ cells upon PTPN13 or β-catenin downregulation. This was confirmed by analysis of the Ki67 proliferation marker, which was significantly reduced in all progenitor subpopulations when compared to control cells. This is in agreement with the increase in quiescence detected in the GFP+ LT-HSCs and GFP+ ST-HSCs. Nevertheless, these observations did not explain why the frequency of HSCs and their quiescence was increased upon downregulation of PTPN13 or β-catenin. Considering the involvement of β-catenin in the cell adhesion process (Valenta et al., 2012), we thought it worthwhile to study whether attachment to the BM was altered. Surprisingly, our in vivo data showed a higher adhesion of mouse progenitor cells to the BM niche upon PTPN13 or β-catenin downregulation. These results are in agreement with many in vitro data that we report showing the alteration of cell adhesion when PTPN13 or β-catenin levels are modified, both in HEL cells and in also in murine hematopoietic progenitor cells. We therefore wondered whether the expression of genes coding for CAMs was altered. The expression of ten different genes, most of them expressed in HSCs (Prowse et al., 2011), was monitored in a cell line downregulated for PTPN13 or β-catenin. Interestingly five of these genes (ITGA4, CDH1, CDH12, NCAM2, and RELN) were upregulated; no differences were found in one of them (ITGA5), and only three of them were downregulated (ITGA11, ITGAL, CDH5). These dramatic changes at the cell surface could ultimately account for the increased adhesiveness. It has been reported previously that β-catenin suppresses the expression of cadherin genes (Jamora et al., 2003). Our analysis of CDH1 gene promoter activity allows us to propose that some of the CAMs coding genes may be β-catenin transcriptional targets, while PTPN13, preserving β-catenin stability (Sardina et al., 2014), would contribute to the transcriptional regulation of these genes. The biology of stem cells depends on their relationship with the niche, and it is clear that CAMs would play a leading role in this respect. For example, it is well known that CDH1 is required for the survival and self-renewal of hESCs (Li et al., 2012), and to maintain HSC multipotency in Drosophila (Gao et al., 2013). Recent reports show that CDH1 overexpression enhances the generation of induced pluripotent stem cells (iPS) (Chen et al., 2010), or the conversion from primed to naive-like pluripotent stem cells (PSCs) (Murayama et al., 2015). Moreover, it has recently been shown that a low expression of Itgal is linked to HSC long-term reconstitution capacity (Fathman et al., 2014).