Upregulations of Gata4 and oxytocin receptor are important in cardiomyocyte differentiation processes of P19CL6 cells

Oxytocin induces P19 cells to differentiate into cardiomyocytes possibly through the oxytocin/oxytocin receptor system. We added oxytocin to the growth medium of P19CL6, a subline of P19, but they did not differentiate into cardiomyocytes as indicated by RT-PCR and Western blotting results. During the cardiac commitment time of P19CL6 cells, the mRNA expression levels of the oxytocin receptor were upregulated by the addition of oxytocin as well as DMSO, but an upregulation of Gata4 expression levels was only observed for the cells induced by DMSO. The in silico analysis of the upstream sequence of the oxytocin receptor predicted putative binding sites for Gata4 and Nkx2.5. These results suggest that upregulations of the oxytocin receptor and Gata4 are important for cardiomyocyte differentiation processes.     

hhlim促进DMSO诱导的P19细胞向心肌分化

为了确定hhlim是否参与胚胎期的心肌分化和发育过程,用可表达hhlim蛋白和hhlim反义RNA的真核表达质粒转染P19胚胎干细胞,经G418筛选得到稳定表达hhlim和hhlim反义RNA的P19细胞克隆后,观察hhlim对P19细胞向心肌分化和发育的影响.结果显示,Nkx2.5和GATA-4在未被外源性hhlim基因转染的P19细胞中不表达.DMSO刺激细胞2天后,GATA-4开始表达,3天后Nkx2.5的表达活性显著升高.hhlim的过表达不但有利于P19细胞的存活和生长,而且还可以使Nkx2.5和GATA-4的表达比对照细胞提前1天.反义hhlim细胞株被DMSO诱导5天后,细胞仍呈集落化生长.同时,Nkx2.5和GATA-4开始表达的时间明显延滞.结果表明,hhlim能促进P19细胞向心肌细胞分化,其作用是通过促进转录因子GATA-4和Nkx2.5的表达而实现的.     

Actin and myosin expression during development of cardiac muscle from cultured embryonal carcinoma cells

P19 embryonal carcinoma cells are multipotential stem cells that differentiate into striated muscle as well as some other cell types when aggregated and exposed to dimethyl sulfoxide (DMSO). Immunofluorescence experiments using monospecific antibodies indicated that the majority of muscle cells were mononucleate and contained four myosin isoforms normally found in cardiac muscle; atrial and ventricular myosin heavy chains, ventricular myosin light chain 1, and atrial myosin light chain 2. Northern blot analysis of RNA isolated from differentiating cultures indicated that cardiac actin and skeletal actin mRNAs were expressed at similar levels and with identical kinetics during the differentiation of P19-derived myocytes. These results demonstrate that most of the P19-derived myocytes are of the cardiac type and suggest that they closely resemble the cells of the early embryonic myocardium.     

The subcellular distribution of protein kinase Calpha, -epsilon, and -zeta isoforms during cardiac cell differentiation.

There is little information on the molecular events that control the subcellular distribution of protein kinase C during cardiac cell differentiation. We examined protein kinase C activity and the subcellular distribution of representatives of the "classical," "novel," and "atypical" protein kinase C's in P19 murine teratoma cells induced to undergo differentiation into cardiac myocytes by the addition of dimethylsulfoxide to the medium (Grepin et al., Development 124, 2387-2395, 1997). Differentiation was assessed by the presence of striated myosin, a morphological marker for cardiac cells. Addition of dimethyl sulfoxide to the medium resulted in the appearance of striated myosin by 10 days postincubation. Immunolocalization and Western blot studies revealed that a significant proportion of protein kinase Calpha, -epsilon, and -zeta were associated with the particulate fraction in P19 cells prior to differentiation. Differentiation into cardiac cells resulted in a translocation of protein kinase C activity from the particulate fraction to cytosol and localization of most of protein kinase Calpha, -epsilon, and -zeta to the cytoplasmic compartment. The total cellular protein kinase C activity was unaltered during differentiation. The translocation of protein kinase C activity during differentiation of P19 cells into cardiac myocytes was associated with a decrease in the levels of cellular 1, 2-diacyl-sn-glycerol. The cellular levels of phosphatidylserine and phosphatidylinositol did not change during differentiation. Addition of 1,2-dioctanoyl-sn-glycerol, a cell-permeant 1, 2-diacyl-sn-glycerol analog, reversed the differentiation-induced switch in the relative distribution of protein kinase C activity and dramatically increased the association of protein kinase Calpha with the particulate fraction. Addition of 1,2-dioctanoyl-sn-glycerol did not reverse the pattern of distribution for protein kinase Cepsilon or -zeta. The results indicate that protein kinase C activity and protein kinase Calpha, -epsilon and -zeta isoforms are redistributed from the particulate to the cytosolic fraction during differentiation of P19 cells into cardiomyocytes. The mechanism for the redistribution of protein kinase Calpha may be related to the reduction in the cellular 1,2-diacyl-sn-glycerol levels that accompany differentiation.     

Expression of metalloproteinases and inhibitors in the differentiation of P19CL6 cells into cardiac myocytes

P19CL6 are a clonal derivative of P19 embryonal carcinoma cells, a euploid, multipotent mouse cell line, that differentiate efficiently into cardiac myocytes, with spontaneous beating evident within 10 days, following DMSO treatment. Using real-time quantitative RT-PCR we have profiled the expression of the complete matrix metalloproteinase and tissue inhibitor of metalloproteinase gene families during P19CL6 differentiation to cardiac myocytes. The genes subdivide into eight groups based upon their expression profile. Their expression was both qualitatively and quantitatively highly homologous to that seen during mouse heart development.     

Combination of retinoic acid,dimethyl sulfoxide and 5-azacytidine promotes cardiac differentiation of human fetal liver-derived mesenchymal stem cells

There are controversial reports about cardiac differentiation potential of mesenchymal stem cells (MSCs), and there is still no well-defined protocol for the induction of cardiac differentiation. The effects of retinoic acid (RA) and dimethyl sulfoxide (DMSO) on the proliferation and differentiation of human fetal liver-derived MSCs (HFMSCs) as well as the pluripotent state induced by 5-azacytidine (5-aza) in vitro were investigated. MSCs were isolated from fetal livers and cultured in accordance with previous reports. Cells were plated and were treated for 24 h by the combination of 5-aza, RA and DMSO in different doses. Different culture conditions were tested in our study, including temperature, oxygen content and medium. Three weeks later, cells were harvested for the certification of cardiac differentiation as well as the pluripotency, which indicated by cardiac markers and Oct4. It was found that the cardiac differentiation was only induced when HFMSCs were treated in the following conditions: in high-dose combination (5-aza 50 μM + RA 10^?1 μM + DMSO 1 %) in cardiac differentiation medium at 37 °C and 20 % O₂. The results of immunohistochemistry and quantitative RT-PCR showed that about 40 % of the cells positively expressed Nkx2.5, desmin and cardiac troponin I, as well as Oct4. No beating cells were observed during the period. The combined treatment with RA, DMSO and 5-aza in high-dose could promote HFMSCs to differentiate into cardiomyocyte-like cells and possibly through the change of their pluripotent state.     

Multiple CArG boxes in the human cardiac actin gene promoter required for expression in embryonic cardiac muscle cells developing in vitro from embryonal carcinoma cells.

Chimeric genes composed of the human cardiac actin promoter driving the Escherichia coli lacZ reporter gene were constructed, transfected, and stably integrated into genomes of P19 embryonal carcinoma cells. The transfected constructs were expressed actively in cardiac myocytes formed following dimethyl sulfoxide (DMSO)-induced cell differentiation but poorly in undifferentiated cultures and in cultures treated with retinoic acid to develop into derivatives of the neuroectoderm. A number of deletions of the promoter were constructed and tested. Three regions required for efficient expression in P19-derived cardiac muscle were identified, each containing sequences referred to as CArG boxes (CC[AT-rich]6GG). This analysis indicated that regulatory sequences important for expression in cardiac muscle were present upstream of the core promoter identified previously by transient assays in skeletal myoblasts. Expression of the cardiac actin promoter was enhanced 10-fold in undifferentiated P19 cells in the presence of the myoD protein. The promoter regions important for expression in P19-derived cardiocytes were similar to those important for myoD-induced enhancement, a result we interpret to be consistent with the idea that cardiac muscle might contain a myoD-like activity.     

Elevation in phosphatidylethanolamine is an early but not essential event for cardiac cell differentiation

The biosynthesis of phosphatidylethanolamine was examined during differentiation of P19 teratocarcinoma cells into cardiac myocytes. P19 cells were induced to undergo differentiation into cardiac myocytes by the addition of dimethyl sulfoxide to the medium. Immunofluorescence labeling confirmed the expression of striated myosin 10 days postinduction of differentiation. The content of phosphatidylethanolamine increased significantly within the first 2 days of differentiation. [1,3-(3)H]Glycerol incorporation into phosphatidylethanolamine was increased 7.2-fold during differentiation, indicating an elevation in de novo synthesis from 1, 2-diacyl-sn-glycerol. The mechanism for the increase in phosphatidylethanolamine levels during cardiac cell differentiation was a 2.8-fold increase in the activity of ethanolaminephosphotransferase, the 1,2-diacyl-sn-glycerol utilizing reaction of the cytidine 5'-diphosphate-ethanolamine pathway of phosphatidylethanolamine biosynthesis. Incubation of P19 cells with the phosphatidylethanolamine biosynthesis inhibitor 8-(4-chlorophenylthio)-cAMP inhibited the differentiation-induced elevation in phosphatidylethanolamine levels but did not affect the expression of striated myosin. The results suggest that elevation in phosphatidylethanolamine is an early event of P19 cell differentiation into cardiac myocytes, but is not essential for differentiation to proceed.