Mechanisms Of Rho GRPase Inhibitors Simvastatin And Y27632on Cardiomvogenesis And Self-renewal Of Mouse Embryonic Stem Cells

Mouse embryonic stem (ES) cell, representing the naive state of pluripotency[1], is known for its ability to self-renew and differentiate in vitro. It is considered as the ideal cell model to study the effect of small molecules for regulation of self-renewal and differentiation. Rho GTPase is a crucial molecular switch that is relevant to multiple aspects of cellular response, such as cytoskeleton regulation, proliferation, cell polarity, gene transcription and so on. Previouse studies have demonstrated the involvement of Rho GTPase in ES cells survival[14-16]. So it is likely that small molecules which influence Rho GTPase might have potential application in ES cell research.So far, most Rho GTPase inhibtors available are indirect inhibitors which block the function of Rho GTPase by targeting protein modification or their downstream effectors[13]. In the present study, Simavastatin (SIM) is a HMG-CoAreductase inhibitor which inhbit Rho GTPases by attenuating prenylation modification of Rho GTPases[17]. Y27632is a downstream kinase inhibor which inhibits Rho associated kinase (ROCK) in RhoA signaling pathway. NSC23766interacts with Racl directly and blocks RhoGEF mediated activation of Racl[18]. In summary, the three chemicals affect different aspects of Rho GTPase signaling and may have distinct effects on ES cells.In this study, we utilized several Rho GTPase inhibitors such as SIM, Y27632and NSC23766to study the role of Rho GTPase in regulating proliferation and cardiomyogenesis of mouse ES cells. The results demonstrated that SIM exhibited significant cardiomyogenenic effect, while Y27632was found to promote proliferation of ES cells. The mechanisms of both chemicals were studied by molecular biology strategies. Moreover, in an effort to find new molecular events that were associated with self-renewal and differentiation, microRNA array analysis and bioinformatice analysis were performed on ES cells, Y-ES cells and EBs, looking for potential targets. 1. Cardiomyogenic effect of SIM on mouse embryonic stem cellObjective:To investigate the role of Rho GTPase on cardiomyocte differentiation of ES cells, and to elucidate the potential mechanism.Results and methods:Western blotting analysis showed that ES cells and EBs expressed the three major Rho GTPases:RhoA, Racl and Cdc42. The effect of Rho GTPase inhibitors were studied by proliferation assay and cardiomyogenesis model of mouse ES cells. The results demonstrate that SIM could promote cardiomyocyte differentiation of ES cells while NSC23766inhibited cardiomyogenesis. Thus it was likely that RhoA and Racl played distinct roles in this process. Further more, we found that SIM promoted cardiomyocte differentiation of mouse ES cells in a dose dependent manner. The concentration of1μM was selected for following experiments. Time coure study found out that a-actinin expression was significantly up-regulated after the4th day of adherent culture. However further experiment showed that SIM treatment at the first3days of adherent culture was also effective in promoting cardiomyocyte differentiation. Moreover, SIM induced cardiomyocytes exhibited mature cardiomyocyte phenotype with clear ladder like sarcomeric structures. In the meantime, western blotting analysis revealed compromised membrane localization of RhoA. Reduced MYPT1phosphrylation was also detected. These results revealed atenuated RhoA signaling in EBs after SIM treatment. Meanwhile, reduced phosphorylation of Focal Adhesion Kinase (FAK) and enhaced activation of Akt was also observed in SIM treated EBs. a-actinin up-regulation was accompanied by a declination in EB Size which was consistant with the previously reported effect of FAK inhibition by PP2.Conclusion:Excessive RhoA signaling hinders cardiomyogenesis of mouse ES cells. SIM promotes cardiomyocyte differentiation of mouse ES cells by reducing membrane location of RhoA, reducing phosphrylation of both MYPT1and FAK. and enhacing Akt activation.2. The effect of Y27632on self-renewal and puripotency regulation of mouse embryonic stem cellsObjective:To investigate the role of ROCK on self-renewal of ES cells and the mechanism involved in the process.Results and methods:Y27632was found to promote ES cell proliferation by MTT assay. Colony forming assay comfirmed the proliferative effect of Y27632. Y27632significanltly increased the number of colonies formed in LIF-withdrawal medium. Moreover, Y27632was also found to support long-term cultivation of mouse ES cells in feeder and LIF free condition. For convenience, Cells cultivated in Y27632medium for a week was identified as Y-ES cells. Western blot and immunostaining experiments revealed that Y-ES cells expressed Pluripotency markers, SSEA1and Oct4similar to ES cell. E-cadherin was finly maitained on the membrane. Gene expression experiments showed that, Y-ES cells expressed similar level of nanog and Oct4with ES cells. GATA4and MMP9, which indicated the differentiation of ES cells, were not detectable in Y-ES cells. However, Rexl, a marker of inner cell mass cells, was down-regulated in Y-ES cells. These results indicated that Y27632could maitain mouse ES cells in a distinct pluripotency state. Further more, in vitro differentiation study demonstrated that Y-ES cells could be successfully differentiated into neural cells, cardiomyocytes and hepatocytes. Teratoma formation test also confirmed that Y-ES cells was able to differentiate into cells derived from all three germ layers. On molecular level, western blotting analysis showed reduced ERK1/2phosphorylation in Y-ES cells. Meanwhile, STAT3phosphorylation was attenuated in Y-ES cells. The results indicated that Y27632induced LIF/STAT independent self-renewal of mouse ES cells. On the other hand TGFβ pathway inhibitor SB431542was able to inhibit self-renewal of Y-ES cells, indicating a possible role for TGFβ pathway in regulating self-renewal of Y-ES cells. MicroRNA array analysis revealed that miR-302b-5p and miR-295-5p, which were reported to be positive regulator of pluripotency, were maintained in Y-ES cells at the similar level with mouse ES cells. Nontheless miR-34b-5p, which was reported to be a negative regulator of pluripotency, was significantly down-regulated in Y-ES cells compared to ES cells.Contusion:ROCK inhibition supports self-renewal of mouse ES cells by inhibiting ERK1/2phosphorylation, E-cadherin relocation, and regulating miRNA expression. Y27632can be used as a substitute for puromycin resistant feeder cells in cultivation of transdected mouse ES cells.3. Bioinformatic analysis of the microRNAs involved in self-renewal of Y-ES cellsObjective:To explore the difference between Y-ES cells and ES cells, and to look for potential pathways that were relevent to self-renewal and differentiation.Results and methods:The differentially expressed miRNAs between Y-ES cells and ES cells were cross checked with previous reported ones between ES cells and epiS cells. Only5miRNAs in27differentialy expressed miRNAs were crossed with those previously reported ones. The result indicated that Y-ES cells may stays in a self-renewing state distinct from both mouse ES cells and epiS cells. To explore the molecular pathways that may be involved in regulating self-renewal of Y-ES cells, functional analysis were performed by miRPath on highly expressed miRNAs in Y-ES cells,8pathways were recognized to be potential targets of those miRNAs. Focal adhesion, Wnt signaling and ErbB signaling was highly involved, it is possible that those pathways might contribute the the difference between Y-ES cells and ES cells. Further more,5miRNAs was confirmed by qRT-PCR to be highly expressed by mouse ES cells and Y-ES cells compared to EB. While another3was conformed to highly expressed by EB. Functional analysis of the two group of miRNAs pointed to13and5targeted pathways. Focal adhesion and ErbB signaling pathway were primarily chosen for protein association analysis. Four targets:Myc, Ctnnbl, Ptk2, GSK3b were indentified as key nodes which were then subjected to western blot analysis. The results demonstrated that Y27632could inhibit the phosphorylation of ERK and GSK3, maitain β-catenin expression and up-regulate c-Myc expression.Conlusion:Y-ES cell states in a distinct selfrenewing state closer to niave state pluripotency.Y27632interferes with the pluripoetncy regulation network via c-Myc, p-GSK, p-ERK, β-catenin may also be involved the self-renewing effect of Y27632.