Canonical NF-κ B Pathway Is Required for Early Mesodermal Differentiation from the Murine Embryonic Stem Cells.
Authors: Tanaka Hirokazu , Matsumura Itaru , Nakao Kazuki , Era Takumi , Kanakura Yuzuru 
Published: April 2020

Abstract
NF-κB family of transcription factors play important roles in cell growth and survival as well as stress and immune responses through the target gene expression. We previously reported that NF-κB family proteins participate in both primitive and definitive hematopoiesis by preventing reactive oxygen species (ROS)-mediated apoptosis. However, their functions in mesodermal development, which is the earlier process to produce hematopoietic cells, has not been elucidated. So, in the present study, we inducibly expressed IκBSR with a Tet-off system in murine embryonic stem (ES) cells and evaluated its effects on mesodermal differentiation. In this system, IκBSR can inhibit the function of canonical NF-κB pathway as a dominant negative mutant in response to tetracycline removal from the culture medium. After 4.0–4.5-day cultures on the OP9 cell layer with differentiation medium deprived of leukemia inhibitory factor (LIF), about 30% of cultured cells developed into the vascular endothelial growth factor receptor 2 (VEGFR2)+ lateral mesodermal cells, which have the potential to differentiate into endothelial cells, hematopoietic cells, and cardiomyocytes. When Tet was deprived from the culture medium, the induced IκBSR inhibited the development of VEGFR2+ cells by inducing apoptosis via the ROS accumulation. However, even if this apoptosis was prevented by the anti-oxidants, the cultured cells did not express VEGFR2. As for this mechanism, we found that although the VEGFR2 promoter was activated by p65, c-Rel, and p50 in response to LIF deprivation in ES cells, IκBSR suppressed this induction almost completely using luciferase assays and ChIP assays. These results suggest that IκBSR blocks the development of lateral mesodermal cells from undifferentiated ES cells through the down-regulated expression of VEGFR2 in concurrence with the induction of apoptosis. To further analyze the effects of IκBSR on mesodermal development, we performed cDNA microarray analysis using differentiated ES cells deprived of Tet for 2 days. Among 4,277 genes, 123 genes were significantly down-regulated and 105 were up-regulated by induction of IκBSR. Among known essential genes for mesodermal development, the expression of platelet derived growth factor receptor α (PDGFRα), which is induced in paraxial mesoderm, was also suppressed by IκBSR significantly. Furthermore, in the prolonged cultures, we found that, although the development of E-cadherin (ECD)+/PDGFRα+ bi-potent mesendodermal cells at day 3.0–3.5 was not affected by IκBSR, it inhibited the subsequent development of ECD−/PDGFRα+ paraxial mesodermal cells at day 5.0–6.0. In accord with this result, we found that IκBSR sustained the expression of Mixl1, one of the intrinsic mesendodermal markers in differentiated ES cells, by semi-quantitive RT-PCR analysis. Together, these results suggest that the canonical NF-κB pathway is required for early mesodermal differentiation from mesendoderm stage through the expression of various essential molecules, thereby regulating their morphogenetic behaviors and cell-fate decisions. We are now evaluating the in vivo effects of IκBSR on the mesodermal development using the tetraploid chimeric mice model, and would like to further disclose the role of NF-κB pathway in our presentation.