The Distinct Signaling Property Of MKK4and MKK7in Mouse Embryonic Stem Cell Differentiation And Developmental Toxicity

Signal transduction pathways are integral components of the developmental regulatory network that guides progressive cell fate determination. The MKK4and MKK7are upstream kinases of the mitogen-activated protein kinases (MAPKs), responsible for channeling physiological and environmental signals to their cellular responses. Both kinases are essential for survival of mouse embryos, but due to embryonic lethality, their precise developmental roles remain largely unknown. In vitro differentiation of mouse embryonic stem cells (ESCs) have opened new possibilities for understanding lineage segregation and gene functioning in the developmental stages that are not normally accessible in vivo. We have developed and validated an experimental system to differentiate mouse ESCs through the formation of embryoid bodies (EBs) in vitro into mesoderm, ectoderm and endoderm lineages while most of the cells into differentiate into mesoderm lineage which focus on cardiomyocyte. Using this system, in combination with gene knockout cell mouse ESCs, we studied the roles of MKK4and MKK7in cardiomyocytes differentiation in vitro. While MKK4and MKK7were dispensable for ESC self-renewal and pluripotency maintenance, they exhibited unique signaling and functional properties in differentiation. MKK4and MKK7complemented each other in activation of the JNK-c-Jun cascades and loss of both led to senescence upon cell differentiation. On the other hand, MKK4and MKK7had opposite effects on activation of the p38cascades during differentiation. Specifically, MKK7reduced p38activation, while Mkk7(-/-) ESCs had elevated phosphorylation of MKK4, p38and ATF2, and increased MEF2C expression. Consequently, the Mkk7(-/-) ESCs had higher expression of MHC and MLC and enhanced formation of contractile cardiomyocytes. In contrast, MKK4was required for p38activation and Mkk4(-/-) ESCs exhibited diminished p-ATF2and MEF2C expression, resulting in impaired MHC induction and defective cardiomyocyte differentiation. Exogenous MKK4expression partially restored the ability of Mkk4(-/-) ESCs to differentiate into cardiomyocytes. Our results uncover complementary and interdependent roles of MKK4and MKK7in development, and identify the essential requirement for MKK4in p38activation and cardiomyocyte differentiation. On the other hand, environmental agents may perturb the MKK4/MKK7signaling to cause developmental toxicity. Application of environmental chemicals to this system led to the identification of nickel and Bisphenol-A as strong toxicants of cardiogenesis. Furthermore, the cardiac toxicity of BPA, but not nickel, was mediated mainly through MKK7.1. Distinct Signaling Properties of Mitogen-activated Protein Kinase Kinases4(MKK4) and7(MKK7) in Embryonic Stem Cell (ESC) Cardiomyocytes Differentiation.(1) Aim:to investigate the possible roles of MAPK pathways in regulating the cardiomyocyte differentiation course of murine embryonic stem (ES)cells in vitro.(2) Methods:the expression of MAPK pathways during cardiomyocytes differentiation was analyzed by using both Western blotting and immunofluorescence. Cardiac specific genes was evaluated at different time courses of ES cell differentiation. The phosphorylation of p38mitogen-activited protein kinase(MAPK) was studied in the differentiation process, and its specific inhibitor SB202190was employed to study the function of P38MAPK on cardiac differentiation. Reconstituton of MKK4was done in the MKK4(-/-) ESCs. The interaction of MKK4- p38was studied by GST-pull down and in vitro kinase assay. The MHC gene regulation by transcription factors was done by chromatin immunopreciptation assay.(3) Results:we developed an experimental system to differentiate mouse ESCs in vitro into mesoderm, ectoderm and endoderm lineages. Using ESCs deficient in the MAP2Ks, we established the MKK4-p38-ATF2and MKK4/7-JNK-c-JUN signaling cascades during differentiation. On the other hand, the MKK4-p38cascade was shown to be essentially required for mesodermal-specific cardiogenesis, while MKK4and MKK7both regulate JNK-c-JUN and differentiating cell survival.(4) Conclusion:Based on these data we suggest that MKK4and MKK7have complemantary and distinct roles in ESC differentiation. The molecular and signaling mechanisms of MAPKs in development can be conveniently elucidated in vitro using ESC culture.2. The roles of MKK7in environmental chemical-caused developmental cardiac toxicity.(1) Aim:to assess the specific contributions of MAPK signaling cascades in environmental chemical-induced cardiac development toxicity.(2) Methods:We treated the ESCs with non-cytotoxic doses of environmental chemicals during in vitro ESCs differentiation and assessed the developmental toxicity by examining gene expression and functional assays. The specific inhibitor of estrogen receptor ICI182,780was employed to study the function of estrogen receptor on BPA and Estrogen caused cardiac development toxcity.(3) Results:Exposure of ESCs to developmental toxicant, the Mkk7(-/-) were not different in response to Ni causing100%inhibition of cardiac foci formation compare to the wild type EBs. Remarkably, the Mkk7(-/-) ESCs showed strong resistance to cardiac toxicity of BPA and Estrogen. Unlike the beating foci in wild type EBs, which was inhibited completely by BPA and Estrogen, those in Mkk7(-/-) were reduced by merely1-2%of the control. This observation suggests that the cardiac toxicity of BPA and Estrogen is mediated largely via MKK7. The specific estrogen receptor inhibitor did not rescue this phenotype, meaning BPAand Estrogen act through estrogen receptor-independent method to cause developmental cardiac toxicity.(4) Conclusion:Based on these data we suggest that MAPKs may be activated by physiological and environmental stimuli and in turn regulate cardiac development. The cardiac toxicity caused by some environmental toxicant may mediated through MKK7signalling pathway.Statistical Analyses:Statistical comparisons were performed using analysis of One-Way ANOVA Dunnett (SPSS13.0).*P values <0.05;**, P values<0.01; and***, P values <0.001are considered significant.